Regenerative polypeptides and uses thereof

ABSTRACT

Described herein are polypeptides comprising an FGF17, IGF2, or BMP7 amino acid sequence and an amino acid sequence from a heterologous polypeptide useful for the treatment of soft-tissue and muscle diseases, disorders, and injuries. Also described herein are synergistic combinations of a Fibroblast Growth Factor Receptor agonist and a glycosaminoglycan, an Insulin-like Growth Factor 1 Receptor (IGF1R) agonist and a short chain fatty acid, and BMP receptor agonists and mTOR activators and/or glycosaminoglycans. Also described are methods of treating muscle and soft-tissue diseases comprising administering the polypeptides and/or synergistic compositions.

CROSS-REFERENCE

This application is a continuation of U.S. application Ser. No.18/163,210 filed Feb. 1, 2023, which is a continuation of U.S.application Ser. No. 17/843,748 filed Jun. 17, 2022, which is a 371filing from PCT application PCT/US2020/066658 filed on Dec. 22, 2020,which claims priority to U.S. provisional application Ser. No.62/953,431 filed Dec. 24, 2019, U.S. provisional application Ser. No.62/953,429 filed Dec. 24, 2019, and U.S. provisional application Ser.No. 62/953,427 filed Dec. 24, 2019, all of which are incorporated byreference in their entirety herein.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in XML format and is hereby incorporated byreference in its entirety. Said copy, created on Jan. 30, 2023, is named“JTI026.xml” and is 186,279 bytes in size.

BACKGROUND

As the average life span increases, increasing emphasis is placed upon“healthy aging.” Individuals would like to live more active lifestylesas they age, and as a result, many aging disorders can have asignificant impact on the quality of life of aging individuals.Treatments directed to regenerative ends have utility for treating agingdiseases. Additionally, many treatments for aging disorders can beapplicable to younger individuals who have suffered illness, injury, orwho possess genetic or developmental defects leading to premature tissueloss, wasting, or weakening.

SUMMARY

Described herein are polypeptides comprising an FGF17, IGF2, or BMP7amino acid sequence and an amino acid sequence from a heterologouspolypeptide useful for the treatment of soft-tissue and muscle diseases,disorders, and injuries. The IGF2 amino acid sequence can include anamino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100%identical to the amino acid sequence set forth in SEQ ID NO: 89. TheBMP7 amino acid sequence can include an amino acid sequence at leastabout 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acidsequence set forth in SEQ ID NO: 89. The BMP7 amino acid sequence caninclude a 15-30 amino acid fragment at least about 90%, 95%, 98%, 99% or100% identical to the amino acid sequence set forth in SEQ ID NO: 93.The FGF17 amino acid sequence can include an amino acid sequence atleast about 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acidsequence set forth in SEQ ID NO: 54. The FGF17 amino acid sequence caninclude a mutation selected from: deletion of amino acids G181-T203,deletion of amino acids 197-T203, deletion of amino acids 204-216,deletion of amino acids 181-216, R204Q/K207Q, deletion of amino acids197-216, K191A/K193A/S200A, and combinations thereof. The FGF17, IGF2,and/or BMP7 can include at least one amino acid that is N-, C-, orO-linked glycosylated.

The heterologous polypeptide can be an immunoglobulin molecule orfragment thereof, an albumin molecule, a transferrin molecule, an XTENsequence, a proline-alanine-serine polymer, a homo-amino acid polymer, aglycine-rich sequence, a gelatin-like polymer, an elastin-like peptide,a carboxy-terminal peptide, or combinations thereof. A fragment of animmunoglobulin molecule can include the hinge domain of an IgG, the CH2domain of an IgG, the CH3 domain of an IgG, or any combination thereof.The immunoglobulin molecule or fragment thereof can include one or moremutations that reduce the effector function of the fragment of theimmunoglobulin molecule. Also described herein are combinations of aFibroblast Growth Factor Receptor agonist and a glycosaminoglycan, anInsulin-like Growth Factor 1 Receptor (IGF1R) agonist and a short chainfatty acid, and BMP receptor agonists and mTOR activators and/orglycosaminoglycans.

Described herein are methods of treating muscle and soft-tissue diseasescomprising administering the polypeptides and/or compositions combininga polypeptide with a small chain fatty acid, mTOR activator, and/orglycosaminoglycan. Muscle diseases that can be treated include, forexample, acute and chronic muscle wasting diseases or conditions, suchas sarcopenia, cachexia, muscular dystrophies, and muscle injury. Softtissue regeneration can be useful to treat acute and chronic musclewasting diseases or conditions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A depicts purified IGF2-hFcm promoted differentiation of humanmyoblast cells.

FIG. 2A depicts sodium butyrate enhanced muscle fusion.

FIG. 2B depicts sodium butyrate enhanced IGF2 activity.

FIG. 2C depicts sodium butyrate enhanced IGF2 activity.

FIG. 3A depicts the change in percent area of eMyHC positive cellstreated with additional doses of vehicle, IGF2, sodium butyrate, or IGF2and sodium butyrate.

FIG. 3B depict the change in percent area of eMyHC positive cellstreated with additional doses of vehicle, IGF2, sodium butyrate, or IGF2and sodium butyrate.

FIG. 4A depicts IGF2 Receptor was expressed on chondrocyte andosteocytes.

FIG. 5A depicts BMP7 induced myoblast proliferation as measured by newlyformed nuclei.

FIG. 5B depicts BMP7 induced myoblast proliferation as measured by totalnuclei.

FIG. 6A depicts leucine enhanced BMP7 mitogenic activity.

FIG. 7A depicts hyaluronic acid (HA) enhanced BMP7 mitogenic activity.

FIG. 8A depicts BMP7 receptors were expressed in human myoblast.

FIG. 9A depicts treatment for chondrocyte proliferation in cartilageinjury and osteoarthritis.

FIG. 10A depicts FGF17-hFcm promoted proliferation of mouse myoblasts asa part of cultured media supernatant, or purified (FIG. 10B).

FIG. 11A depicts FGF17 sequence mutations improved protein expressionlevels in CHO cells.

FIG. 11B depicts FGF17 sequence mutations promoted proliferation ofmouse myoblasts as a part of cultured media supernatant, or purified.

FIG. 12A depicts additional FGF17 mutants improved protein expressionlevels in CHO cells.

FIG. 12B depicts additional FGF17 sequence mutations promotedproliferation of mouse myoblasts as a part of cultured mediasupernatant, or purified.

FIG. 13A depicts FGF17 receptor was expressed in human myoblasts.

FIG. 14A depicts heparin enhanced FGF17 mitogenic activity.

FIG. 15A depicts hyaluronic acid (HA) enhanced FGF17 mitogenic activity.

FIG. 16A depicts an experimental overview that demonstratedintramuscular administration of FGF17 promoted the regeneration ofmuscle in BaCl2 injured old mice model.

FIG. 16B depicts intramuscular administration of FGF17 promoted theregeneration of muscle in BaCl2 injured old mice model as measured bynew fiber formation.

FIG. 16C depicts intramuscular administration of FGF17 promoted theregeneration of muscle in BaCl2 injured old mice model by reducingfibrosis.

FIG. 17A depicts an experimental overview that demonstrated systemicadministration of FGF17 protects against Dexamethasone induced muscleatrophy.

FIG. 17B-D depicts systemic administration of FGF17 protected againstDexamethasone induced muscle atrophy as measured by percent muscle masschange (FIG. 17B), forelimb specific force (FIG. 17C) and bothlimb force(FIG. 17D).

DETAILED DESCRIPTION

In certain aspects disclosed herein is a therapeutically active proteinor polypeptide sequence or derivative or fragment thereof that enhancesprogenitor cell growth or regeneration or function through activation ofa cell surface receptor, and one or more of: a secretion signal amultimerizing component, or a stabilizing component. We modify andcombined the sequences of certain polypeptides to create secreted,therapeutically active proteins with applications to muscle and softtissue regeneration useful to treat acute and chronic muscle wastingdiseases or conditions, such as sarcopenia, cachexia, musculardystrophies, and muscle injury. In certain aspects, disclosed herein isa method of treating individuals with acute and chronic muscle wastingdiseases or conditions, such as sarcopenia, cachexia, musculardystrophies, and muscle injury.

In certain aspects, disclosed herein is a polypeptide comprising an FGF8subfamily amino acid sequence and a heterologous polypeptide amino acidsequence, wherein the heterologous polypeptide increases the stabilityor biological function of the FGF8 subfamily amino acid sequence. Incertain aspects, disclosed herein is a composition comprising an FGFRagonist and a glycosaminoglycan.

In certain aspects, disclosed herein is a polypeptide comprising an IGF2amino acid sequence and a heterologous polypeptide amino acid sequence,wherein the heterologous polypeptide amino acid sequence increases thestability or biological function of the IGF2 amino acid sequence. Incertain aspects, disclosed herein is a composition comprising an IGF1Ragonist and a short fatty acid chain.

In certain aspects, disclosed herein is a polypeptide comprising a BMP7amino acid sequence and a heterologous polypeptide amino acid sequence,wherein the heterologous polypeptide increases the stability orbiological function of the BMP7 amino acid sequence. In certain aspects,disclosed herein is a composition comprising a BMP7 receptor agonist anda glycosaminoglycan.

The secretion signal sequence can either be one naturally occurring witha therapeutically active protein or polypeptide sequence or a differentone selected, modified, or created to optimize expression yield throughsecretion efficiency, processing kinetics, or cell line specificprocessing. Further examples and SEQ IDs are in Table 1. In certainaspects, the polypeptide may comprise a secretory signal peptide. Incertain embodiments, the secretory signal peptide is one of SEQ ID NO:10-16. Production of the fusion polypeptides may be done in heterologousproduction systems (e.g., bacteria, yeast, mammalian, inset, etc.).

Polypeptides can induce a regenerative effect through membrane receptorsin desired cell types. Examples from the stem cell secretome selectedfor their ability to improve muscle and soft tissue regeneration arelisted in Table 2, and include, for example, FGF17, BMP7, IGF2, andvariants thereof. Multimerizing components can join two or more otherprotein components together. A multimerizing component can take the formof a linker sequence of amino acids that joins other components tandemlyinto a single consecutive amino acid sequence. Or multimerizingcomponents can take the form of proteins or protein domains thatdimerize, resulting in covalent disulfide linking or non-covalentlyassociations driving dimerization. Examples are found in Table 3.

Stabilizing components can reduce degradation, increase translational orpost-translation folding, reduce unfolding rates, increase half-life,and/or improve other desirable pharmacokinetic parameters (e.g., serumhalf-life, Cmax, AUC, Tmax, etc.). Examples can include abundant,circulating proteins or fragments thereof such as albumin or thefragment crystallizable (Fc) region from a human antibody. Furtherexamples are in Table 3.

Certain Definitions

In the following description, certain specific details are set forth inorder to provide a thorough understanding of various embodiments.However, one skilled in the art will understand that the embodimentsprovided may be practiced without these details. Unless the contextrequires otherwise, throughout the specification and claims whichfollow, the word “comprise” and variations thereof, such as, “comprises”and “comprising” are to be construed in an open, inclusive sense, thatis, as “including, but not limited to.” As used in this specificationand the appended claims, the singular forms “a,” “an,” and “the” includeplural referents unless the content clearly dictates otherwise. Itshould also be noted that the term “or” is generally employed in itssense including “and/or” unless the content clearly dictates otherwise.Further, headings provided herein are for convenience only and do notinterpret the scope or meaning of the claimed embodiments.

As used herein the term “about” refers to an amount that is near thestated amount by 10%.

As used herein the terms “individual,” “patient,” or “subject” are usedinterchangeably and refer to individuals diagnosed with, suspected ofbeing afflicted with, or at-risk of developing at least one disease forwhich the described compositions and method are useful for treating. Incertain embodiments the individual is a mammal. In certain embodiments,the mammal is a mouse, rat, rabbit, dog, cat, horse, cow, sheep, pig,goat, llama, alpaca, or yak. In certain embodiments, the individual is ahuman.

As used herein the term “treat” or “treating” refers to interventions toa physiological or disease state of an individual designed or intendedto ameliorate at least one sign or symptom associated with saidphysiological or disease state. The skilled artisan will recognize thatgiven a heterogeneous population of individuals afflicted with adisease, not all individuals will respond equally, or at all, to a giventreatment.

As used herein, the term “heterologous” refers to a nucleotide or aminoacid sequence that is from a different source (e.g., gene, polypeptide,or organism) compared to the amino acid or nucleotide sequence to whichit refers to as being heterologous. Heterologous includes biologicalsequences derived from different organisms or to sequences derived fromdifferent sources (e.g., genes or proteins) of the same organism.Heterologous sequences include recombinant DNA molecules comprisingnucleotide sequences from different sources, fusion proteins comprisingamino acid sequences from different sources, and epitope or purificationtags of natural or synthetic origin.

As used herein, the term “muscle” refers to skeletal muscle, and doesnot refer to smooth muscle or cardiac muscle.

As used herein, the term “soft tissue” refers to connective tissues,including without limitations, tendons, ligaments, and cartilage.

As used herein, the term “mitogenic activity” refers to an activity thatinduces cell division or proliferation.

As used herein, the term “fusion promoting activity” refers to activitythat promotes the fusion of cells into multinucleated cells, such as thefusion of myocytes into multinucleated myofibers, or advances thedifferentiation of a terminal differentiating stem or progenitor cellstoward a committed cell lineage type, such as the progression ofmyoblasts into myocytes or the increase in cell size of expandingmyofibers.

The terms “polypeptide” and “protein” are used interchangeably to referto a polymer of amino acid residues, and are not limited to a minimumlength. Polypeptides, including the provided antibodies and antibodychains and other peptides, e.g., linkers and binding peptides, mayinclude amino acid residues including natural and/or non-natural aminoacid residues. The terms also include post-expression modifications ofthe polypeptide, for example, glycosylation, sialylation, acetylation,phosphorylation, and the like. In some aspects, the polypeptides maycontain modifications with respect to a native or natural sequence, aslong as the protein maintains the desired activity. These modificationsmay be deliberate, as through site-directed mutagenesis, or may beaccidental, such as through mutations of hosts which produce theproteins or errors due to PCR amplification.

Percent (%) sequence identity with respect to a reference polypeptidesequence is the percentage of amino acid residues in a candidatesequence that are identical with the amino acid residues in thereference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that areknown for instance, using publicly available computer software such asBLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Appropriateparameters for aligning sequences are able to be determined, includingalgorithms needed to achieve maximal alignment over the full length ofthe sequences being compared. For purposes herein, however, % amino acidsequence identity values are generated using the sequence comparisoncomputer program ALIGN-2. The ALIGN-2 sequence comparison computerprogram was authored by Genentech, Inc., and the source code has beenfiled with user documentation in the U.S. Copyright Office, WashingtonD.C., 20559, where it is registered under U.S. Copyright RegistrationNo. TXU510087. The ALIGN-2 program is publicly available from Genentech,Inc., South San Francisco, Calif., or may be compiled from the sourcecode. The ALIGN-2 program should be compiled for use on a UNIX operatingsystem, including digital UNIX V4.0D. All sequence comparison parametersare set by the ALIGN-2 program and do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows: 100 times thefraction X/Y, where X is the number of amino acid residues scored asidentical matches by the sequence alignment program ALIGN-2 in thatprogram's alignment of A and B, and where Y is the total number of aminoacid residues in B. It will be appreciated that where the length ofamino acid sequence A is not equal to the length of amino acid sequenceB, the % amino acid sequence identity of A to B will not equal the %amino acid sequence identity of B to A. Unless specifically statedotherwise, all % amino acid sequence identity values used herein areobtained as described in the immediately preceding paragraph using theALIGN-2 computer program.

The polypeptides described herein can be encoded by a nucleic acid. Anucleic acid is a type of polynucleotide comprising two or morenucleotide bases. In certain embodiments, the nucleic acid is acomponent of a vector that can be used to transfer the polypeptideencoding polynucleotide into a cell. As used herein, the term “vector”refers to a nucleic acid molecule capable of transporting anothernucleic acid to which it has been linked. One type of vector is agenomic integrated vector, or “integrated vector,” which can becomeintegrated into the chromosomal DNA of the host cell. Another type ofvector is an “episomal” vector, e.g., a nucleic acid capable ofextra-chromosomal replication. Vectors capable of directing theexpression of genes to which they are operatively linked are referred toherein as “expression vectors.” Suitable vectors comprise plasmids,bacterial artificial chromosomes, yeast artificial chromosomes, viralvectors and the like. In the expression vectors regulatory elements suchas promoters, enhancers, polyadenylation signals for use in controllingtranscription can be derived from mammalian, microbial, viral or insectgenes. The ability to replicate in a host, usually conferred by anorigin of replication, and a selection gene to facilitate recognition oftransformants may additionally be incorporated. Vectors derived fromviruses, such as lentiviruses, retroviruses, adenoviruses,adeno-associated viruses, and the like, may be employed. Plasmid vectorscan be linearized for integration into a chromosomal location. Vectorscan comprise sequences that direct site-specific integration into adefined location or restricted set of sites in the genome (e.g.,AttP-AttB recombination). Additionally, vectors can comprise sequencesderived from transposable elements.

FGF17 Polypeptides

In certain aspects, described herein, are FGF17 polypeptides thatcomprise an FGF17 amino acid sequence. The FGF17 amino acid sequence canbe a human FGF17. The FGF17 amino acid sequence can have at least about90%, 95%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 54. TheFGF17 amino acid sequence can be 100% identical to SEQ ID NO: 54. TheFGF17 amino acid sequence can be at least about 90%, 95%, 97%, 98%, 99%,or 100% identical to SEQ ID NO: 55. The FGF17 amino acid sequence can be100% identical to SEQ ID NO: 55. The FGF17 amino acid sequence can be atleast about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 56.The FGF17 amino acid sequence can be 100% identical to SEQ ID NO: 56.The FGF17 amino acid sequence can be at least about 90%, 95%, 97%, 98%,99%, or 100% identical to SEQ ID NO: 57, wherein the sequence comprisesthe R204Q and K207Q mutations. The FGF17 amino acid sequence can be 100%identical to SEQ ID NO: 57. The FGF17 amino acid sequence can be atleast about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 58.The FGF17 amino acid sequence can be 100% identical to SEQ ID NO: 58.The FGF17 amino acid sequence can be at least about 90%, 95%, 97%, 98%,99%, or 100% identical to SEQ ID NO: 59, wherein the sequence comprisesthe K191A, K193A, and S200A mutations. The FGF17 amino acid sequence canbe 100% identical to SEQ ID NO: 59.

The FGF17 polypeptides described herein can be fusion proteins orpolypeptides that may comprise additional heterologous (non-FGF17) aminoacid sequences that enhance the expression, stability or function of theFGF17 polypeptide. These heterologous amino acid sequences may increasethe expression of the FGF17 fusion polypeptide from a cell system (e.g.,CHO cells or other suitable cell system for bulk production) by 10%,20%, 25%, 30%, 40%, 50%, 75%, 100%, 150%, 200%, 200%, 400%, 500%, 1,000%or more compared to a polypeptide not comprising the heterologous aminoacid sequence. These heterologous amino acid sequences may increase thebioavailability (e.g., increasing the T1/2) of the FGF17 polypeptide invivo by 10%, 20%, 25%, 30%, 40%, 50%, 75%, 100%, 150%, 200%, 200%, 400%,500%, 1,000% or more compared to a polypeptide not comprising theheterologous amino acid sequence. These heterologous amino acidsequences may increase the function (e.g., signaling through an FGFreceptor) of the FGF17 polypeptide in vivo by 10%, 20%, 25%, 30%, 40%,50%, 75%, 100%, 150%, 200%, 200%, 400%, 500%, 1,000% or more compared toa polypeptide not comprising the heterologous amino acid sequence.

The FGF17 amino acid sequence of the FGF17-heterologous polypeptidefusion protein can be at least about 90%, 95%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 54. The FGF17 amino acid sequence of the fusionprotein can be 100% identical to SEQ ID NO: 54. The FGF17 amino acidsequence of the fusion protein can be at least about 90%, 95%, 97%, 98%,99%, or 100% identical to SEQ ID NO: 55. The FGF17 amino acid sequenceof the fusion protein can be 100% identical to SEQ ID NO: 55. The FGF17amino acid sequence of the fusion protein can be at least about 90%,95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 56. The FGF17 aminoacid sequence of the fusion protein can be 100% identical to SEQ ID NO:56. The FGF17 amino acid sequence of the fusion protein can be at leastabout 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 57,wherein the sequence comprises the R204Q and K207Q mutations. The FGF17amino acid sequence of the fusion protein can be 100% identical to SEQID NO: 57. The FGF17 amino acid sequence of the fusion protein can be atleast about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 58.The FGF17 amino acid sequence of the fusion protein can be 100%identical to SEQ ID NO: 58. The FGF17 amino acid sequence of the fusionprotein can be at least about 90%, 95%, 97%, 98%, 99%, or 100% identicalto SEQ ID NO: 59, wherein the sequence comprises the K191A, K193A, andS200A mutations. The FGF17 amino acid sequence of the fusion protein canbe 100% identical to SEQ ID NO: 59.

An FGF17 fusion polypeptide amino acid sequence can be at least about90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 60, SEQ ID NO:61, or SEQ ID NO: 62. The fusion polypeptide amino acid sequence can beat least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:65, wherein the sequence comprises the R204Q and K207Q mutations. Thefusion polypeptide amino acid sequence can be 100% identical to SEQ IDNO: 66, SEQ ID NO: 66 or SEQ ID NO: 71, wherein the sequence comprisesthe K191A, K193A, and S200A mutations. The fusion polypeptide amino acidsequence can be 100% identical to SEQ ID NO: 72. The fusion polypeptideamino acid sequence can be at least about 90%, 95%, 97%, 98%, 99%, or100% identical to SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, or SEQ IDNO: 69, wherein the sequence comprises the R204Q and K207Q mutations.

The FGF17 amino acid sequence can be at least about 80%, 90%, 95%, 97%,98%, 99% or 100% identical to one of SEQ ID NO: 54-70 or 74, with 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 amino acids are deleted from the N- and/orC-terminus of the polypeptide.

IGF2 Fusion Proteins

Described herein are certain therapeutically useful IGF2 polypeptides,including IGF2 fusion polypeptides that promote in vivo stability andfunction of the IGF2 comprising polypeptides.

In certain aspects described herein are IGF receptor ligandpolypeptides. The IGF2 polypeptides can comprise an IGF2 amino acidsequence. The IGF2 amino acid sequence can be that of a human IGF2polypeptide. The human IGF2 polypeptide can comprise amino acids 25 to91 of SEQ ID NO. 79 (i.e. SEQ ID NO. 76). The IGF2 amino acid sequencecan be at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to oneof SEQ ID NO. 76, 79, 81 or 86. The IGF2 amino acid sequence can be 100%identical to SEQ ID NO. 76. The IGF2 amino acid sequence can be at leastabout 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or 100% identical to one ofSEQ ID NO: 76, 79-81, 86, or 88 and 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10amino acids are deleted from the N- and/or C-terminus of thepolypeptide.

In certain IGF2 polypeptides described herein are fusion proteins orpolypeptides that may comprise additional heterologous (non-IGF2) aminoacid sequences that enhance the expression, stability or function of theIGF2 polypeptide compared to a polypeptide not comprising theheterologous amino acid sequence. These heterologous amino acidsequences may increase the expression of the IGF2 fusion polypeptidefrom a cell system (e.g., CHO cells or other suitable cell system forbulk production) by 10%, 20%, 25%, 30%, 40%, 50%, 75%, 100%, 150%, 200%,200%, 400%, 500%, 1,000% or more compared to a polypeptide notcomprising the heterologous amino acid sequence. These heterologousamino acid sequences may increase the bioavailability or otherpharmacokinetic factor (e.g., increasing the T1/2, AUC, Cmax, Tmax,etc.) of the IGF2 polypeptide in vivo by 10%, 20%, 25%, 30%, 40%, 50%,75%, 100%, 150%, 200%, 200%, 400%, 500%, 1,000% or more compared to apolypeptide not comprising the heterologous amino acid sequence. Theseheterologous amino acid sequences may improve the pharmacodynamicsand/or increase the function (e.g., signaling through an IGF receptor)of the IGF2 polypeptide in vivo by 10%, 20%, 25%, 30%, 40%, 50%, 75%,100%, 150%, 200%, 200%, 400%, 500%, 1,000% or more compared to apolypeptide not comprising the heterologous amino acid sequence.

Also described herein are IGF receptor ligand fusion polypeptides orpolypeptides that include an amino acid sequence heterologous to IGF2.The IGF receptor ligand fusion includes a heterologous amino acidsequence that promotes the stability or function of the IGF receptorligand. The IGF2 amino acid sequence of the IGF2-heterologouspolypeptide fusion protein can be at least about 90%, 95%, 97%, 98%,99%, or 100% identical to SEQ ID NO. 76. The IGF2 amino acid sequence ofthe fusion protein can be 100% identical to SEQ ID NO. 76. The IGF2amino acid sequence of the fusion protein can be at least about 90%,95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO. 80. The IGF2 aminoacid sequence of the fusion protein can be 100% identical to SEQ ID NO.80. The IGF2 amino acid sequence of the fusion protein can be at leastabout 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO. 88. TheIGF2 amino acid sequence of the fusion protein can be 100% identical toSEQ ID NO. 88. Additional representative sequences can be found in Table2.

BMP7 Fusion Proteins

Described herein are certain therapeutically useful BMP7 polypeptides,including BMP7 fusion polypeptides that promote in vivo stability andfunction of the BMP7 comprising polypeptides.

In one aspect, described herein, are BMP7 polypeptides, that comprise aBMP7 amino acid sequence. The BMP7 amino acid sequence can be a humanBMP7 amino acid sequence. The BMP7 amino acid sequence can comprise orconsist of amino acids 293 to 431 of BMP7. The BMP7 amino acid sequencecan comprise a BMP7 knuckle domain (SEQ ID NO: 92). The BMP7 sequencecan be at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQID NO: 89. The BMP7 sequence can be 100% identical to SEQ ID NO: 89. TheBMP7 sequence can be at least about 90%, 95%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 92. The BMP7 sequence can be 100% identical toSEQ ID NO: 92. The BMP7 polypeptide sequence can comprise one, two,three, four or more repeats of a BMP7 knuckle domain.

The BMP7 amino acid sequence may be further fused to a heterologousamino acid sequence, either directly or with a linker sequence betweenthe BMP7 amino acid sequence and the heterologous polypeptide amino acidsequence to create a fusion polypeptide. The fusion polypeptide cancomprise a human BMP7 amino acid sequence. The fusion polypeptide cancomprise or consist of amino acids 293 to 431 of BMP7. The fusionpolypeptide can comprise a BMP7 knuckle domain (SEQ ID NO: 92). Thefusion polypeptide amino acid sequence can comprise a BMP7 amino acidsequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical toSEQ ID NO: 89. The fusion polypeptide can comprise a BMP7 amino acidsequence 100% identical to SEQ ID NO: 89. The fusion polypeptide aminoacid can comprise a BMP7 amino acid sequence at least about 90%, 95%,97%, 98%, 99%, or 100% identical to SEQ ID NO: 92. The fusionpolypeptide can comprise a BMP7 amino acid sequence 100% identical toSEQ ID NO: 92. The fusion polypeptide amino acid sequence can be atleast about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 90.The fusion polypeptide amino acid sequence can be 100% identical to SEQID NO: 90. The fusion polypeptide amino acid sequence can be at leastabout 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 91. Thefusion polypeptide amino acid sequence can be 100% identical to SEQ IDNO: 91.

The BMP7 amino acid sequence can be at least about 80%, 90%, 95%, 97%,98%, 99% or 100% identical to one of SEQ ID NO: 20, 21, 32, or 89, with1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids deleted from the N- and/orC-terminus of the polypeptide.

In some aspects, the BMP7 sequence may be a fragment of a BMP7 sequence.The knuckle domain of BMP7 comprises amino acids 98 to 129 of SEQ ID NO:89 (SEQ ID NO: 32). 15-30 amino acid fragments from the knuckle domaincan activate BMP signaling. The BMP7 sequence can comprise a knuckledomain of BMP. The BMP7 sequence can be a 15-30 amino acid fragment ofSEQ ID NO: 32. The BMP7 sequence can be at least about 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 29, 29, 30, 31, 32, 33, 34,or 35 amino acids of SEQ ID NO: 32.

Secretory Signal Peptides

In certain aspects, the fusion polypeptide may comprise a secretorysignal peptide. The secretory signal peptide can be SEQ ID NO. 24, SEQID NO 25, SEQ ID NO 26, SEQ ID NO 27, SEQ ID NO 28, SEQ ID NO 29, or SEQID NO: 30. Production of the fusion polypeptides herein in heterologousproduction systems (e.g., bacteria, yeast, mammalian, insect, etc.) mayinvolve the use of an appropriate secretory signal sequence for theparticular host cell.

Multimerizing components join two or more other protein components. Amultimerizing component may comprise a linker sequence of amino acidsthat joins other components that are identical or different into asingle consecutive amino acid sequence. Suitable linkers includepolypeptide linkers such as a Gly-Ser linker or spacer described herein.A multimerizing component can take the form of proteins or proteindomains that multimerize or dimerize, resulting in covalent disulfidelinking (e.g., through the addition of one or more de novo cysteineresidues) or non-covalent associations driving dimerization (e.g., aleucine zipper). The multimerizing components may link or multimerize aplurality of IGF2 amino acid sequences. The multimerizing components maylink or multimerize two IGF2 amino acid sequences. The two IGF2 aminoacid sequences may be the same, or different, and selected from any ofthe IGF2 sequences described herein. The multimerizing components maylink or multimerize two, three, four, five or more IGF2 amino acidsequences. The multimerizing components may link or multimerize an IGF2amino acid sequence with another polypeptide that provides fusionpromoting, proliferation promoting function, increased plasma half-life,or improvement of other pharmacokinetic or pharmacodynamic parameters.

The FGF17, IGF2, or BMP7 amino acid sequence may comprise functionalfragments, mutated sequences, or modified polypeptides thereof. Table 2lists some exemplary fragments, polypeptides and modified polypeptides.The IGF2 or BMP7 sequence can be N-, C-, or O-linked glycosylated. TheIGF2 sequence can be glycosylated at one amino acid. The IGF2 sequencecan be glycosylated at a site corresponding to Thr96, Thr99, or Thr163of SEQ ID NO. 31. The BMP7 sequence can comprise at least oneglycosylated amino acid. The BMP7 can be glycosylated at residues Asn10,Asn29, or Asn90 of SEQ ID NO. 89.

The FGF17, IGF2, or BMP7 receptor ligand polypeptides and receptorligand fusion polypeptides described herein may be encoded by nucleicacids to facilitate production of the receptor ligand polypeptide orfusion polypeptide. These nucleic acids can be compatible withbacterial, yeast, insect, or mammalian expression systems. They maycomprise promoters/enhancers (either constructive or inducible),polyadenylation signals, selectable markers (such as antibioticresistance), origins of replication or other accessory nucleic acidsequences. FGF17, IGF2, or BMP7 sequences can be used from manyorganisms. The FGF17, IGF2, or BMP7 sequence can comprise a human FGF17,IGF2, or BMP7 amino acid sequence. The FGF17, IGF2, or BMP7 sequence cancomprise a cat, dog or a horse FGF17, IGF2, or BMP7 sequence. The FGF17,IGF2, or BMP7 sequence can comprise a mouse, rat, rabbit, dog, cat,horse, cow, sheep, pig, goat, llama, alpaca, yak, or monkey sequence.

FGF17 Nucleic Acid Sequences

In certain embodiments, the FGF17 nucleic acid sequence is at leastabout 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO. 17. TheFGF17 nucleic acid sequence can be 100% identical to SEQ ID NO. 17. TheFGF17 nucleic acid sequence is at least about 90%, 95%, 97%, 98%, 99%,or 100% identical to SEQ ID NO. 22. The FGF17 nucleic acid sequence canbe 100% identical to SEQ ID NO. 22. The FGF17 nucleic acid sequence canbe at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ IDNO. 23. The FGF17 nucleic acid sequence can be 100% identical to SEQ IDNO. 23.

IGF2 Nucleic Acid Sequences

The IGF2 nucleic acid sequence can be at least about 90%, 95%, 97%, 98%,99%, or 100% identical to SEQ ID NO. 39. The IGF2 nucleic acid sequencecan be 100% identical to SEQ ID NO. 39. The IGF2 nucleic acid sequencecan be at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQID NO. 43. The IGF2 nucleic acid sequence can be 100% identical to SEQID NO. 43. The IGF2 nucleic acid sequence can be at least about 90%,95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO. 46. The IGF2 nucleicacid sequence can be 100% identical to SEQ ID NO. 46.

BMP7 Nucleic Acid Sequences

The BMP7 nucleic acid sequence can be at least about 90%, 95%, 97%, 98%,99%, or 100% identical to SEQ ID NO: 51. The BMP7 nucleic acid sequencecan be 100% identical to SEQ ID NO: 51. The BMP7 nucleic acid sequencecan be at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQID NO: 52. The BMP7 nucleic acid sequence can be 100% identical to SEQID NO: 52. The BMP7 nucleic acid sequence can be at least about 90%,95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 53. The BMP7 nucleicacid sequence can be 100% identical to SEQ ID NO: 53.

Heterologous Peptides

The heterologous polypeptide that comprises part of the fusion proteinsdescribed herein may comprise, consist, or consist essentially of afragment of an immunoglobulin molecule, an albumin molecule, atransferrin molecule, an XTEN sequence, a proline-alanine-serinepolymer, a homo-amino acid polymer, a glycine-rich sequence, agelatin-like polymer, an elastin-like peptide, a carboxy-terminalpeptide, or combinations thereof.

In one aspect described herein the therapeutic polypeptide is FGFreceptor ligand polypeptide or an FGF17 polypeptide. In one aspectdescribed herein the therapeutic polypeptide is IGF receptor ligandpolypeptide or an IGF2 polypeptide. In one aspect described herein thetherapeutic polypeptide is BMP receptor ligand polypeptide or an BMP7polypeptide.

In one aspect described herein the therapeutic polypeptide fused to aheterologous polypeptide amino acid sequence, either directly or througha linker, wherein the heterologous amino acid sequence imparts increasedfunction or stability to the therapeutic polypeptide.

The heterologous peptide can improve the pharmacokinetics,pharmacodynamics, stability or biological function of the therapeuticamino acid sequence. The heterologous sequence may be fused to thetherapeutic amino acid sequence at the C-terminus or at the N-terminusof the therapeutic amino acid sequence. The therapeutic amino acidsequence can be fused to a heterologous sequence at the N-terminus. Thetherapeutic amino acid sequence can be fused to a heterologous sequenceat the C-terminus. A flexible linker can be used between the therapeuticamino acid sequence and the heterologous sequence at the N terminus. Aflexible linker can be used between the therapeutic amino acid sequenceand the heterologous sequence at the C terminus. A spacer can be usedbetween the therapeutic amino acid sequence and the heterologoussequence at the N terminus. A spacer can be used between the therapeuticamino acid sequence and the heterologous sequence at the C terminus.

Heterologous peptides can improve the pharmacokinetics,pharmacodynamics, stability, or the biological function of the IGF2amino acid sequence. Fusion proteins can be used to improve thepharmacokinetics of the biologically active molecules, such as byprolonging the half-life, as discussed in Strohl, “Fusion Proteins forHalf-Life Extension of Biologics as a Strategy to Make Biobetters,”BioDrugs (2015) 29:215-239. Fusing a polypeptide to a molecule or afragment of a molecule with a long half-life, such as an immunoglobulin,an albumin, or a transferrin increase the half-life of the polypeptide.An XTEN sequence is a repeating amino acid polymer containing the aminoacid residues A, E, G, P, S, and T which when fused to a peptide iscapable of extending the half-lives of the peptides, while beingotherwise inert. Fusing small repeating sequences such asproline-alanine-serine polymers (repeats of proline, alanine andserine), a homo-amino acid polymer sequence such as glycine-richsequences (G-G-G-S), gelatin-like proteins, and elastin-like sequences(V-P-G-x-G, where x is any amino acid except proline) can also extendthe half-life of a polypeptide. Fusing a polypeptide to acarboxy-terminal peptide (CTP) can increase the half-life of thepolypeptide in the serum due to the strong negative change of CTP. Theheterologous polypeptide can comprise a fragment of an immunoglobulinmolecule, an albumin molecule, a transferrin molecule, an XTEN sequence,a proline-alanine-serine polymer, a homo-amino acid polymer, aglycine-rich sequence, a gelatin-like polymer, an elastin-like peptide,a carboxy-terminal peptide, or combinations thereof.

Immunoglobulins are large effector molecules and, for example, IgGimmunoglobulins have a plasma half-life of approximately 21 days. Whenan immunoglobulin fragment is fused to second polypeptide, this canincrease the half-life of the second polypeptide. The fragment of theimmunoglobulin molecule can comprise the hinge domain of an IgG, the CH2domain of an IgG, the CH3 domain of an IgG, or any combination thereof.The fragment of the immunoglobulin molecule can comprise the hingedomain of IgG1, the CH2 domain of IgG1, the CH3 domain of IgG1, or anycombination thereof. The fragment of the immunoglobulin molecule cancomprise the hinge domain of IgG4, the CH2 domain of IgG4, the CH3domain of IgG4, or any combination thereof.

In some circumstances, mutations of the immunoglobulin molecule orfragment may increase the half-life or stability of the immunoglobulinmolecule or fragment. The fragment of the immunoglobulin molecule cancomprise the hinge domain of IgG1, the CH2 domain of IgG1, the CH3domain of IgG1, or any combination thereof with one or more of thefollowing amino acid mutations in the immunoglobulin molecule: P329G,L234A and L235A. The fragment of the immunoglobulin molecule comprisesan IgG4 molecule. The fragment of the immunoglobulin molecule cancomprise an IgG4 molecule with at least one of the following amino acidmutations in the immunoglobulin molecule: N434A, N434H,T307A/E380A/N434A, M252Y/S254T/T256E, 433K/434F/436H, T250Q, T250F,M428L, M428F, T250Q/M428L, N434S, V308W, V308Y, V308F, M252Y/M428L,D2591/V308F, M428L/V308F, Q311V/N434S, T307Q/N434A, E258F/V427T, S228P,L235E, S228P/L235E/R409K, S228P/L235E, K370Q, K370E, deletion of G446,deletion of K447, and combinations thereof of IgG4 according to the EUnumbering system.

Secretory signal sequences are sequence motifs that target proteins tothe secretory pathway in the cell. Secretory sequences may be cleavedfrom the protein to produce the mature, secreted protein. Thepolypeptide can comprise a secretory signal sequence. The polypeptidecan comprise human FGF17, IGF2, or BMP7 secretory sequence (SEQ ID NO 9,SEQ ID NO 11, SEQ ID NO 12). The polypeptide can comprise a secretorysignal that is SEQ ID NO. 10, SEQ ID. NO. 13, SEQ ID NO. 14, SEQ ID NO.15, or SEQ ID NO. 16.

Linkers and Spacers

Linkers or spacers are short amino acid sequences that separatedifferent domains in a single protein, or domains between fusionproteins. As used herein, the term “linker” and spacer” areinterchangeable. Linkers can either be rigid or flexible. Rigid linkersmay prevent unwanted interactions between different domains.Proline-rich linkers tend to be more rigid, while glycine rich linkerstend to be more flexible. Flexible linkers may allow domains within asingle protein to interact. Another use for flexible linkers is tocovalently bond protein complexes and binding partners to generatestable protein complexes. Flexible linkers may also be used to promotedimerization. Linkers and spacers are reviewed in Chichili et al,Linkers in the Structural biology of protein-protein interactions,Protein Sci. February 2013. 22(2): 153-167.

The fusion polypeptides described herein may further comprise a linkeror a spacer amino acid sequence that separate the therapeuticspolypeptide and the heterologous polypeptide. The linker or spacer canbe a peptide linker or spacer. The linker or spacer can be a flexiblelinker or spacer. The linker can be three alanines (AAA). The peptidelinker can be a glycine-serine linker. The linker can be (in one-letteramino acid code): GGGGS (4GS) or multimers of the 4GS linker, such asrepeats of 2, 3, 4, or 5 4GS linkers. The glycine-serine linker cancomprise the amino acid sequence set forth in SEQ ID NO: 94 or 95, or 2,3, 4, 5, or repeats of SEQ ID NO: 94 or 95. The linker can comprise atleast 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32 amino acidsderived from neither the polypeptide sequences in Table 2 nor theheterologous polypeptide amino acid sequences of Table 3.

The linker or spacers can be a single amino acid residue or greater inlength. In certain embodiments, the peptide linker is at least 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, or 32 amino acids in length. The peptidelinker can have at least one amino acid residue but is no more than 32,31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14,13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acid residues in length.

Combinations of FGFR Agonists and Glycosaminoglycans

In certain aspects, disclosed herein is a composition comprising an FGFRagonist and a glycosaminoglycan. There are four FGF receptors, FGF1R,FGFR2, FGFR3, FGFR4, which are expressed by a variety of tissuesthroughout the body. Chemical agonists of FGFR include withoutlimitations, PF-05231023 and SUN11602. Other polypeptides, includingdekafin and hexafins, are also capable of activating FGFR signaling.These compositions can comprise an unexpected synergistic effect and areuseful for treating the muscle and/or soft-tissue conditions ordisorders. This synergistic effect may also be promoted by methodscomprising separate administration of an FGFR agonist and aglycosaminoglycan. The combinations described herein can impartadditional treatment utility and function to each of the individualcomponents of the combination.

FGFR1 can be activated by FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF8,FGF10, FGF17, FGF19, FGF20, FGF21, FGF22, and FGF23. The FGFR1 agonistcan be an FGFR1 agonistic antibody, an FGF polypeptide or a functionalfragment thereof, FGF17 or a functional fragment thereof, PF-05231023,SUN11602, a dekafin, a hexafin, or combinations thereof.

FGFR2 comprises two alternatively spliced isoforms. FGFR2IIIb binds toFGF1, FGF3, FGF10 and FGF22, while FGFR2IIIc binds to FGF1, FGF2, FGF4,FGF6, FGF8, FGF9, FGF17, and FGF18. The FGFR2 agonist can be an FGFR2agonistic antibody, an FGF polypeptide or a functional fragment thereof,FGF17 or a functional fragment thereof, PF-05231023, SUN11602, adekafin, a hexafin, or combinations thereof.

FGFR3 can be activated by at least FGF1, FGF2, FGF4, FGF5, FGF6, FGF8,FGF9, FGF16, FGF17, FGF18, FGF20, FGF9, FGF19, FGF21, and FGF23, andFGF17. Mutations in FGFR3 have been associated with defects inchrondrocyte proliferation and calcification, as well as achondroplasia.The FGFR3 agonist can be an FGFR3 agonistic antibody, an FGF polypeptideor a functional fragment thereof, FGF17 or a functional fragmentthereof, PF-05231023, SUN11602, a dekafin, a hexafin, or combinationsthereof.

FGFR4 can be activated by at least FGF1, FGF2, FGF4, FGF6, FGF7, FGF8,FGF9, FGF16, FGF17 and FGF18. The FGFR4 agonist can be an FGFR4agonistic antibody, an FGF polypeptide or a functional fragment thereof,FGF17 or a functional fragment thereof, PF-05231023, SUN11602, adekafin, a hexafin, or combinations thereof.

The FGFR agonist can be a member of the FGF8 subfamily. The FGFR agonistcan be an FGFR1 agonist. The FGFR agonist can be FGF17. The FGF17polypeptide can be at least about 90%, 95%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 54. The FGF17 can be 100% identical to SEQ IDNO: 54. The FGF17 polypeptide can be at least about 90%, 95%, 97%, 98%,99%, or 100% identical to SEQ ID NO: 55. The FGF17 polypeptide can be100% identical to SEQ ID NO: 55. The FGF17 polypeptide can be at leastabout 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 56. TheFGF17 can be 100% identical to SEQ ID NO: 56. The FGF17 polypeptide canbe at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ IDNO: 57, wherein the sequence comprises the R204Q and K207Q mutations. Incertain embodiments, the FGF17 polypeptide is 100% identical to SEQ IDNO: 57. In certain embodiments, the FGF17 polypeptide is at least about90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 58. The FGF17polypeptide can be 100% identical to SEQ ID NO: 58. The FGF17polypeptide can be at least about 90%, 95%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 59, wherein the sequence comprises the K191A,K193A, and S200A mutations.

The FGF17 polypeptide can be 100% identical to SEQ ID NO: 59.

Glycosaminoglycans are linear polysaccharides containing repeatingdisaccharide units. There are four glasses of glycosaminoglycans:heparin/heparin sulfate, chondroitin sulfate/dermatan sulfate, keratinsulfate, and hyaluronic acid. The glycosaminoglycan can be aheparin/heparin sulfate, a chondroitin sulfate/dermatan sulfate, akeratin sulfate, or a hyaluronic acid. In some embodiments, theglycosaminoglycan comprises a heparin. The glycosaminoglycan cancomprise a hyaluronic acid.

Heparin can be derived from natural sources, often referred to asunfractionated heparin. Heparin can also be defined based upon molecularweight. Low molecular weight heparin includes dalteparin, enoxaparin,certoparin, ardeparin, parnaparin, reviparin, nadroparin, anddanaparoid. Heparin can be administered in a mixture with othercompounds, such as danaparoid, which is a mixture of heparan sulfate,dermatan sulfate, and chrondriotin sulfate. The composition can compriselow molecular weight heparin, heparin sulfate, unfractionated heparin,heparin tetrasaccharide, dalteparin, tinzaparin, enoxaparin, certoparin,ardeparin, parnaparin, reviparin, nadroparin, heparin flush, danaparoid,fondaparinux, or combinations thereof.

Hyaluronic acid (HA) is a polymeric molecule and can exhibit a range ofmolecular weights. Hyaluronic acid can be used at almost any average ofmodal molecular weight formulation of The molecular weight can be, forexample, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500,1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700,2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900,4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000 kDa ormore, or any range derivable therein. HA can include low molecularweight HA (about 500 to 700 kilodaltons kDa), medium molecular weight HA(700-1000 kDa), and high molecular weight HA (1.0-4.0 million daltons(MDa)). HA includes natural formulations, synthetic formulations, orcombinations thereof. In some embodiments, the HA is a low molecularweight, a medium molecular weight, a low molecular weight, or acombination thereof. In some embodiments, the HA is a natural HA, asynthetic HA, or a combination thereof.

The HA may be a hyaluronic acid derivative. Examples of chemicalmodifications which may be made to HA include any reaction of an agentwith the four reactive groups of HA, namely the acetamido, carboxyl,hydroxyl, and the reducing end. HA derivatives include, withoutlimitations, hydrophobized hyaluronan, maleimide modified HA,methacrylated hyaluronic acid, or a sulfated hyaluronic acid. In someembodiments, the HA is modified at an acetamido group, a carboxyl group,a hydroxyl group, a reducing end, or combinations thereof. The HA cancomprise a hydrophobized hyaluronan, a maleimide modified HA, amethacrylated hyaluronic acid, a sulfated hyaluronic acid, or acombination thereof. The HA can be covalently cross-linked via proteinsor organic molecules into higher molecular weight moieties.

Also described herein are methods comprising administering an FGFRagonist and a glycosaminoglycan. The administration can be in the samecomposition, separate formulations. When separate formulations areadministered they can be administered effectively simultaneously (e.g.,during the same treatment) or separately with an interval of at least 1hour, 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7days, or more. When separate formulations are administered they can beadministered by the same route or different routes selected fromintravenous, intradermal, and subcutaneous. The formulations, whetherseparate or singular can be administered directly to the site of muscleor soft-tissue injury.

Combinations of an IGF1R Agonist and a Short Fatty Acid Chain

In certain aspects, disclosed herein is a composition comprising anIGF1R agonist and a short fatty acid chain. IGF1R signaling activatesdownstream pathways including pathways involved in cell proliferation,cell differentiation, and cell survival. The two IGF ligands, IGF1 andIGF2, activate IGF1R signaling. Additional peptides that activate IGF1Rsignaling are INS. Other agonists of IGF1R include, without limitations,demethylasterriquinone B1, Ginsenoside Rg5, and the human antimicrobialpeptide LL-37. T can he IGF1R agonist comprise an IGF1R agonisticantibody, an IGF polypeptide or a functional fragment thereof, IGF2 or afunctional fragment thereof, insulin, demethylasterriquinone B1,Ginsenoside Rg5, LL-37, or combinations thereof. These compositionscomprise an unexpected synergistic effect and are useful for treatingthe muscle and/or soft-tissue conditions or disorders. This synergisticeffect may also be promoted by methods comprising separateadministration of an IGF1R agonist and a short fatty acid chain.

The IGF2R agonist can be an IGF ligand. The IGF1R agonist can be IGF2.The IGF2 polypeptide can be at least about 90%, 95%, 97%, 98%, 99%, or100% identical to SEQ ID NO. 76. The IGF2 polypeptide can be 100%identical to SEQ ID NO. 76. The IGF2 polypeptide can be at least about90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO. 80. The IGF2polypeptide can be 100% identical to SEQ ID NO. 80. The IGF2 polypeptidecan be at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQID NO. 81. The IGF2 polypeptide can be 100% identical to SEQ ID NO. 81.

The composition can comprise an IGF1R agonist and a short fatty acidchain. Short fatty acid chains include, without limitations, butyrates,a phenylbutyrate, valproic acid, propionic acid, methanoic acid,ethanoic acid, 2-methylpropanoic acid, 3-methylbutanoic acid, pentanoicacid, and a multimerized version thereof such as tributyrin. Butyratesinclude, without limitations, butyric acids, sodium butyrate, methylbutyrate, ethyl butyrate, butyl butyrate, pentyl butyrate, or sodiumbutyrate. The short chain fatty acid can be a butyrate. The butyrate canbe butyric acid. The butyrate can be sodium butyrate. The short chainfatty acid can be a phenylbutyrate, valproic acid, propionic acid,methanoic acid, ethanoic acid, 2-methylpropanoic acid, 3-methylbutanoicacid, pentanoic acid, or a multimerized version thereof such astributyrin.

Also described herein are methods comprising administering an IGF1Ragonist and a short fatty acid chain. The administration can be in thesame composition, separate formulations. When separate formulations areadministered, they can be administered effectively simultaneously (e.g.,during the same treatment) or separately with an interval of at least 1hour, 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7days, or more.

Combinations of BMP Receptor Agonists and mTOR Activators

In certain aspects, disclosed herein is a composition comprising a BMPreceptor agonist and an mTOR activator. BMP receptors activatedownstream signaling through the TGF-beta pathway and are involved inmany cell functions including differentiation, proliferation, andmigration. There are two classes of BMP receptors: BMP type I (ACVR1,BMPR1A, and BMPR1B), and BMP type II (BMP2R, ACVR2A, and ACVR2B). BMPtype 1 receptors bind BMP ligands exclusively, while BMP type IIreceptors bind BMPs and related proteins, including activin, Gdf9, andGDf11. The BMP receptor agonist can comprise an ACVR1 agonist, a BMPR1Aagonist, a BMPR1B agonist, a BMP2R agonist, an ACVR2A agonist, an ACVR2Bagonist, or a combination thereof. The BMP receptor agonist can comprisean ACVR1 agonist, an ACVR2A agonist, an ACVR2B agonist, a BMPR1Aagonist, or a combination thereof. The BMP receptor can comprise anACVR1 agonist. The BMP receptor agonist can comprise an ACVR2A agonist.The BMP receptor can comprise an ACRV2B agonist. The BMP receptor cancomprise a BMPR1A agonist. The BMP receptor agonist can comprise anACVR1 agonist antibody, a BMPR1A agonist antibody, a BMPR1B agonistantibody, a BMP2R agonist antibody, a ACVR2A agonist antibody, a ACVR2Bagonist antibody, a BMP polypeptide or a functional fragment thereof, aBMP7 or a functional fragment thereof, ventromorphin, SB4, tacrolimus,isoliquiritigeni, alantolactone, PD407824, or combinations thereof.These compositions comprise an unexpected synergistic effect and areuseful for treating the muscle and/or soft-tissue conditions ordisorders. This synergistic effect may also be promoted by methodscomprising separate administration of a BMP receptor agonist and aleucine.

The BMP7 polypeptide can be at least about 90%, 95%, 97%, 98%, 99%, or100% identical to SEQ ID NO: 89. The BMP7 polypeptide can be 100%identical to SEQ ID NO: 89. The BMP7 polypeptide can be at least about90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 90. The BMP7polypeptide can be 100% identical to SEQ ID NO: 90. The BMP7 polypeptidecan be at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQID NO: 91. The BMP7 polypeptide can be 100% identical to SEQ ID NO: 91.The BMP7 polypeptide can be at least about 90%, 95%, 97%, 98%, 99%, or100% identical to SEQ ID NO: 93. The BMP7 polypeptide can be 100%identical to SEQ ID NO: 93.

The mammalian target of rapamycin (mTOR) pathway is a key regulator ofskeletal muscle mass and growth. mTOR is a serine/threonine kinaseinvolved in diverse cellular processes including cell growth,differentiation, autophagy, survival, and metabolism. mTOR activationthrough the mTORC1 complex is required both for myofibrillar muscleprotein synthesis and skeletal muscle hypertrophy. Inactivation of themTORC1 complex has been found to be associated with loss of muscle massand muscle struct during muscle wasting due to old age, cachexia, andatrophy due to physical activity.

The mTOR signaling pathway can be activated by many different signals,including amino acids, polypeptides, and small molecules. The mTORactivator can be an amino acid. The amino acid can be leucine, valine,isoleucine or a combination thereof. The amino acid can be leucine. ThemTOR activator can be a polypeptide. The polypeptide can comprise a Rashomolog enriched in brain (Rheb), tuberous sclerosis complex (TSC),protein kinase B (PKB), extracellular-signal-regulated kinase 1/2(ERK1/2), p90 ribosomal s6 kinase 1 (RSK1), Wnt ligands, or acombination thereof. The mTOR activator can be a small molecule. ThemTOR activator can comprise MHY1485, NV-5138,3-benzyl-5-((2-nitrophenoxy)methyl)-dihydrofuran-2(3H)-one (3BDO), or acombination thereof. The mTOR activator can comprise leucine, valine,isoleucine, Ras homolog enriched in brain (Rheb), tuberous sclerosiscomplex (TSC), protein kinase B (PKB), extracellular-signal-regulatedkinase 1/2 (ERK1/2), p90 ribosomal s6 kinase 1 (RSK1), a Wnt ligand,MHY1485, NV-5138,3-benzyl-5-((2-nitrophenoxy)methyl)-dihydrofuran-2(3H)-one (3BDO), or acombination thereof.

The mTOR activator can comprise leucine. The amino acid leucine is animportant part of mTOR signaling within skeletal muscle. Leucine is abranched chain amino acid that is essential to the human diet. It is thesingle most common amino acid in human proteins, at a frequency ofnearly 1 in 10 amino acids (UniProtKB/Swiss-Prot release 2013_04—April2013). The circulating and intracellular concentrations of this aminoacid are monitored and tightly controlled as part of feedback mechanismscontrolling major anabolic and catabolic processes such as celldivision, protein synthesis, and autophagy. Part of the regulatoryeffects of leucine are mediated by the mTORC1 complex whose activationto drive protein synthesis and cell cycle progression are in part drivenby intracellular leucine concentration. The combination of a BMPreceptor agonist and leucine or another branched chain amino aciddemonstrate synergistical mitogenic activity.

Synthetic leucine derivatives may be used. The leucine can comprise1-leucine, glycyl-1-leucine, acetyl-1-leucine, l-leucine ethyl ester,and 1-leucine methyl ester, caproic acid, phthaloyl-1-leucine,benzoyl-dl-leucine, or a combination thereof. The leucine can be a salt.The leucine can comprise 1-leucenium hydrogen maleate, leucinehydrochloride, or a combination thereof.

Also described herein are methods comprising administering a BMPreceptor agonist and an mTOR activator. The administration can be in thesame composition, or separate formulations. When separate formulationsare administered, they can be administered effectively simultaneously(e.g., during the same treatment) or separately with an interval of atleast 1 hour, 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6days, 7 days, or more.

Therapeutic Indications

In certain aspects, the fusion polypeptides comprising an FGF8 subfamilyamino acid sequence and a heterologous polypeptide, compositionscomprising an FGFR agonist and a glycosaminoglycan, and the methodsdescribed herein, are useful for treating diseases and disorders thatinvolve soft-tissue injury, degradation, or destruction, or for use intreating an individual with an aging disorder, a muscle wastingdisorder, a muscle injury, an injury to a connective tissue, or aninjury to a non-muscle soft-tissue, or any combination thereof.

In certain aspects, the fusion polypeptides comprising an IGF ligandamino acid sequence and a heterologous polypeptide, compositionscomprising an IGF1R agonist and a short fatty acid chain, and themethods described herein, are useful for treating diseases and disordersthat involve soft-tissue injury, degradation, or destruction, or for usein treating an individual with an aging disorder, a muscle wastingdisorder, a muscle injury, an injury to a connective tissue, or aninjury to a non-muscle soft-tissue, or any combination thereof.

In certain aspects, the fusion polypeptides comprising a BMP7 amino acidsequence, compositions comprising a BMP receptor agonist and aglycosaminoglycan, compositions comprising a BMP receptor agonist and anmTOR activator, and the methods, described herein, are useful fortreating diseases and disorders that involve soft-tissue injury,degradation, or destruction, or for use in treating an individual withan aging disorder, a muscle wasting disorder, a muscle injury, an injuryto a connective tissue, or an injury to a non-muscle soft-tissue, or anycombination thereof.

Aging disorders that result in the deterioration and loss of muscletissue are such disorders. Sarcopenia, for example, is the degenerativeloss of skeletal muscle mass quality, and strength and can be associatedwith aging. Injuries that result in acute muscle damage are other muscledisorders, which are treatable by the polypeptides, compositions andmethods described herein. The disorders include muscle ruptures,strains, and contusions. A rupture is a separating of the muscletissues. Muscle strains are contraction-induced injuries in which musclefibers tear due to extensive mechanical stress, and can be classified asa grade I, II, or III. Muscle contusions are muscle hematomas. Muscleinjury can also be caused by non-mechanical stresses such as cachexia.Cachexia may be caused by malnutrition, cancer, AIDS, coeliac disease,chronic obstructive pulmonary disease, multiple sclerosis, rheumatoidarthritis, congestive heart failure, tuberculosis, familial amyloidpolyneuropathy, mercury poisoning (acrodynia), Crohn's disease,untreated/severe type 1 diabetes mellitus, anorexia nervosa,chemotherapy, muscular dystrophy or other genetic diseases which causeimmobility, and hormonal deficiencies. Certain disorders that areweaknesses of specific muscles such as dysphagia or facioscapulohumeralmuscular dystrophy may also be treated by the polypeptides describedherein. Additional soft-tissues disorders that may be treated using thepolypeptides comprising an FGF8 subfamily amino acid sequence and/orcompositions comprising an FGFR agonist and a glycosaminoglycandescribed herein are those that inflict injury to the tendons, ligamentsor cartilage. Additional soft-tissues disorders that may be treatedusing the polypeptides comprising an IGF ligand amino acid sequence andcompositions comprising an IGF1R agonist and a short fatty acid chaindescribed herein are those that inflict injury to the tendons, ligamentsor cartilage. Additional soft-tissues disorders that may be treatedusing the fusion polypeptides comprising a BMP7 amino acid sequence,compositions comprising a BMP receptor agonist and a glycosaminoglycan,and the methods, described herein, are those that inflict injury to thetendons, ligaments or cartilage.

The muscle wasting disease can be a muscular dystrophy. The musculardystrophy can comprise a myotonic muscular dystrophy, Duchenne musculardystrophy, Becker muscular dystrophy, Limb-girdle muscular dystrophy,facioscapulohumeral muscular dystrophy, congenital, muscular dystrophy,oculopharyngeal muscular dystrophy, or distal muscular dystrophy. Themuscular dystrophy can be myotonic dystrophy.

The aging disorder can be sarcopenia. The muscle wasting disorder can becachexia. The cachexia can be a result of a cancer, AIDS, end stagekidney disease, or cardiovascular disease. The injury can be a muscleinjury. The muscle wasting can be atrophy due to limb immobilization ordisuse. The muscle injury can be a strain or a tear. The muscle injurycan be a Grade III strain. Sarcopenia can contribute to the incidence ofthe muscle injury. The injury can be ligament damage. The ligamentdamage can be a rupture or a tear. The injury can be tendon damage. Thetendon damage can be a rupture or a tear. The injury can be cartilagedamage.

The compositions described herein, are for use in a method of treatingmyositis. The myositis can comprise dermatomyositis, polymyositis,necrotizing myopathy (also called necrotizing autoimmune myopathy orimmune-mediated necrotizing myopathy), juvenile myositis, or sporadicinclusion-body myositis.

The compositions described herein can be for use in a method of treatingcartilage related-disorders. The cartilage related disorder may be dueto tears, injuries, or wear. The cartilage-associated disease may beosteoarthritis, osteochondritis dissecans, achondroplasia, ordegenerative cartilage lesions.

The compositions described herein can be for use in a method ofincreasing proliferation or promoting survival of a cell associated withsoft-tissue damage. The polypeptides comprising an IGF ligand amino acidsequence and compositions comprising an IGF1R agonist and a short fattyacid chain described herein can be useful in a method of increasingproliferation or promoting survival of any one or more of a muscle cell,a muscle precursor cell, a tenocyte, a tenocyte precursor cell, achondrocyte, a chondrocyte precursor cell, a mesenchymal stem cell, or afibroblast.

Muscle fibrosis is an excessive accumulation of extracellular matrixcomponents, including collagen. Muscle fibrosis impairs muscle function,negatively affects muscle regeneration after injury, and increasesmuscle susceptibility to re-injury. The compositions described hereincan be for use in a method of reducing muscle fibrosis. The fibrosis canbe associated with aging, muscular dystrophy, or an injury. The IGFligand can be IGF2.

In order to differentiate into mature muscle cells, myoblasts must fuseand form multinucleated cells. In certain embodiments, the fusionpolypeptides comprising an IGF ligand amino acid sequence and aheterologous polypeptide, compositions comprising an IGF1R agonist and ashort fatty acid chain, and the methods described herein are for use ina method of increasing myoblast fusion. The IGF ligand can be IGF2.

The fusion polypeptides comprising an IGF ligand amino acid sequence anda heterologous polypeptide, compositions comprising an IGF1R agonist anda short fatty acid chain, and the methods described herein can be foruse in a method of increasing muscle mass. Muscle mass can be increasedby at least about 1%, 2.5%, 5%, 10%, 20%, 30%, 40%, 50% or more than50%. The IGF ligand can be IGF2.

The fusion polypeptides comprising an IGF ligand amino acid sequence anda heterologous polypeptide, compositions comprising an IGF1R agonist anda short fatty acid chain, and the methods described herein can be foruse in a method of increasing grip strength. Grip strength can beincreased by at least about 1%, 2.5%, 5%, 10%, 20%, 30%, 40%, 50% ormore than 50%. The IGF ligand can be IGF2.

The fusion polypeptides comprising an IGF ligand amino acid sequence anda heterologous polypeptide, compositions comprising an IGF1R agonist anda short fatty acid chain, and the methods described herein can be foruse in a method of increasing muscle endurance. Muscle endurance can beincreased by at least about 1%, 2.5%, 5%, 10%, 20%, 30%, 40%, 50% ormore than 50%. The IGF ligand can be IGF2.

Methods of Treatment

In certain aspects, disclosed herein is a method of treating anindividual with a disorder comprising administering an FGFR agonist anda glycosaminoglycan to the individual with the disorder. The FGFRagonist and the glycosaminoglycan can be administered in separateformulations. The FGFR agonist and the glycosaminoglycan can beadministered simultaneously. The FGFR agonist and the glycosaminoglycancan be administered at different times. The glycosaminoglycan can be aheparin. The glycosaminoglycan can be a hyaluronic acid.

In certain aspects, disclosed herein is a method of treating anindividual with a disorder comprising administering an FGFR1 agonist anda glycosaminoglycan to the individual the disorder. The FGFR1 agonistand the glycosaminoglycan (e.g., heparin) can be administered inseparate formulations. The FGFR1 agonist and the glycosaminoglycan(e.g., heparin) can be administered simultaneously. The FGFR1 agonistand the glycosaminoglycan (e.g., heparin) can be administered atdifferent times. The glycosaminoglycan can be a heparin. Theglycosaminoglycan can be a hyaluronic acid.

In certain aspects, disclosed herein is a method of treating anindividual with a disorder comprising administering a polypeptidecomprising a FGF8 subfamily amino acid sequence and a glycosaminoglycan(e.g., heparin) or a compound or mixture comprising a glycosaminoglycanto the individual the disorder. The polypeptide comprising a FGF8subfamily amino acid sequence and the glycosaminoglycan (e.g., heparin)can be administered in separate formulations. The polypeptide comprisinga FGF8 subfamily amino acid sequence and the glycosaminoglycan (e.g.,heparin) can be administered simultaneously. The polypeptide comprisinga FGF8 subfamily amino acid sequence and the glycosaminoglycan (e.g.,heparin) can be administered at different times. The glycosaminoglycancan be a hyaluronic acid.

In certain aspects, disclosed herein is a method of treating anindividual with a disorder comprising administering a polypeptidecomprising a FGF17 amino acid sequence and a glycosaminoglycan (e.g.,heparin) or a compound or mixture comprising glycosaminoglycan to theindividual the disorder. The polypeptide comprising a FGF17 amino acidsequence and the glycosaminoglycan (e.g., heparin) can be administeredin separate formulations. The polypeptide comprising a FGF17 amino acidsequence and the glycosaminoglycan (e.g., heparin) can be administeredsimultaneously. The polypeptide comprising a FGF17 amino acid sequenceand the glycosaminoglycan can be administered at different times. Theglycosaminoglycan can be a hyaluronic acid.

In certain aspects, disclosed herein is a method of treating anindividual with a disorder comprising administering a polypeptidecomprising an FGF8 subfamily amino acid sequence. The polypeptide cancomprise a FGF17 amino acid sequence. The polypeptide can comprise aFGF17 fusion protein.

In certain aspects, disclosed herein is a method of treating anindividual with a disorder comprising administering an IGF1R agonist anda short fatty acid chain (e.g., butyrate) to the individual. The IGF1Ragonist and the short fatty acid chain (e.g., butyrate) can beadministered in separate formulations. The IGF1R agonist and the shortfatty acid chain (e.g., butyrate) can be administered simultaneously.The IGF1R agonist and the short fatty acid chain (e.g., butyrate) can beadministered at different times.

In certain aspects, disclosed herein is a method of treating anindividual with a disorder comprising administering a polypeptidecomprising an IGF ligand amino acid sequence and a butyrate to theindividual the disorder. The polypeptide comprising the IGF ligand aminoacid sequence and the butyrate can be administered in separateformulations. The polypeptide comprising the IGF ligand amino acidsequence and the butyrate can be administered simultaneously. Thepolypeptide comprising the IGF ligand amino acid sequence and thebutyrate can be administered at different times.

In certain aspects, disclosed herein is a method of treating anindividual with a disorder comprising administering a polypeptidecomprising an IGF2 amino acid sequence and a short fatty acid chain(e.g., butyrate) to the individual the disorder. The polypeptidecomprising the IGF ligand amino acid sequence and the short fatty acidchain (e.g., butyrate) can be administered in separate formulations. Thepolypeptide comprising the IGF2 amino acid sequence and the short fattyacid chain (e.g., butyrate) can be administered simultaneously. Thepolypeptide comprising the IGF2 amino acid sequence and the short fattyacid chain (e.g., butyrate) can be administered at different times.

In certain aspects, disclosed herein is a method of treating anindividual with a disorder comprising administering a BMP receptoragonist and a glycosaminoglycan to the individual the disorder. The BMPreceptor agonist and a glycosaminoglycan can be administered in separateformulations. The BMP receptor agonist and a glycosaminoglycan can beadministered simultaneously. The BMP receptor agonist and aglycosaminoglycan can be administered at different times. Theglycosaminoglycan can be a heparin. The glycosaminoglycan can be ahyaluronic acid.

In certain aspects, disclosed herein is a method of treating anindividual with a disorder comprising administering a polypeptidecomprising a BMP7 amino acid sequence and hyaluronic acid or a compoundor mixture comprising hyaluronic acid to the individual the disorder.The polypeptide comprising a BMP7 amino acid sequence and the hyaluronicacid are administered in separate formulations. In some embodiments, thepolypeptide comprising a BMP7 amino acid sequence and the hyaluronicacid are administered simultaneously. In some embodiments, thepolypeptide comprising a BMP7 amino acid sequence and the hyaluronicacid are administered at different times.

In certain aspects, disclosed herein is a method of treating anindividual with a disorder comprising administering a polypeptidecomprising a BMP7 amino acid sequence and heparin or a compound ormixture comprising heparin to the individual the disorder. In someembodiments, the polypeptide comprising a BMP7 amino acid sequence andthe heparin are administered in separate formulations. In someembodiments, the polypeptide comprising a BMP7 amino acid sequence andthe heparin are administered simultaneously. In some embodiments, thepolypeptide comprising a BMP7 amino acid sequence and the heparin areadministered at different times.

In certain aspects, disclosed herein is a method of treating anindividual with a disorder comprising administering a BMP receptoragonist and an mTOR activator to the individual the disorder. In someembodiments, the BMP receptor agonist and an mTOR activator areadministered in separate formulations. In some embodiments, the BMPreceptor agonist and an mTOR activator are administered simultaneously.In some embodiments, the BMP receptor agonist and an mTOR activator areadministered at different times. In certain embodiments, mTOR activatoris a leucine.

In certain aspects, disclosed herein is a method of treating anindividual with a disorder comprising administering a polypeptidecomprising a BMP7 amino acid sequence and leucine or a compound ormixture comprising leucine to the individual the disorder. In someembodiments, the polypeptide comprising a BMP7 amino acid sequence andthe leucine are administered in separate formulations. In someembodiments, the polypeptide comprising a BMP7 amino acid sequence andthe leucine are administered simultaneously. In some embodiments, thepolypeptide comprising a BMP7 amino acid sequence and the leucine areadministered at different times.

In certain aspects, disclosed herein is a method of treating anindividual with a disorder comprising administering a BMP receptoragonist and a glycosaminoglycan to the individual the disorder. In someembodiments, the BMP receptor agonist and a glycosaminoglycan areadministered in separate formulations. In some embodiments, the BMPreceptor agonist and a glycosaminoglycan are administeredsimultaneously. In some embodiments, the BMP receptor agonist and aglycosaminoglycan are administered at different times. In certainembodiments, the glycosaminoglycan is a heparin. In certain embodiments,the glycosaminoglycan is a hyaluronic acid.

In certain aspects, disclosed herein is a method of treating anindividual with a disorder comprising administering a polypeptidecomprising a BMP7 amino acid sequence and a glycosaminoglycan (e.g.,heparin or hyaluronic acid) or a compound or mixture comprising aglycosaminoglycan (e.g., heparin or hyaluronic acid) to the individualthe disorder. In some embodiments, the polypeptide comprising a BMP7amino acid sequence and the glycosaminoglycan (e.g., heparin orhyaluronic acid) are administered in separate formulations. In someembodiments, the polypeptide comprising a BMP7 amino acid sequence andthe glycosaminoglycan (e.g., heparin or hyaluronic acid) areadministered simultaneously. In some embodiments, the polypeptidecomprising a BMP7 amino acid sequence and the glycosaminoglycan (e.g.,heparin or hyaluronic acid) are administered at different times.

The treatment can be administered by any suitable route such as, forexample, subcutaneous, intravenous, or intramuscular. In certainembodiments, the treatment is administered on a suitable dosageschedule, for example, weekly, twice weekly, monthly, twice monthly,once every three weeks, or once every four weeks. The treatment can beadministered in any therapeutically effective amount. Thetherapeutically effective amount can be about 0.001 mg/kg to about 1mg/kg. The therapeutically effective amount can be about 0.001 mg/kg toabout 0.002 mg/kg, about 0.001 mg/kg to about 0.005 mg/kg, about 0.001mg/kg to about 0.01 mg/kg, about 0.001 mg/kg to about 0.02 mg/kg, about0.001 mg/kg to about 0.05 mg/kg, about 0.001 mg/kg to about 0.1 mg/kg,about 0.001 mg/kg to about 0.2 mg/kg, about 0.001 mg/kg to about 0.5mg/kg, about 0.001 mg/kg to about 1 mg/kg, about 0.002 mg/kg to about0.005 mg/kg, about 0.002 mg/kg to about 0.01 mg/kg, about 0.002 mg/kg toabout 0.02 mg/kg, about 0.002 mg/kg to about 0.05 mg/kg, about 0.002mg/kg to about 0.1 mg/kg, about 0.002 mg/kg to about 0.2 mg/kg, about0.002 mg/kg to about 0.5 mg/kg, about 0.002 mg/kg to about 1 mg/kg,about 0.005 mg/kg to about 0.01 mg/kg, about 0.005 mg/kg to about 0.02mg/kg, about 0.005 mg/kg to about 0.05 mg/kg, about 0.005 mg/kg to about0.1 mg/kg, about 0.005 mg/kg to about 0.2 mg/kg, about 0.005 mg/kg toabout 0.5 mg/kg, about 0.005 mg/kg to about 1 mg/kg, about 0.01 mg/kg toabout 0.02 mg/kg, about 0.01 mg/kg to about 0.05 mg/kg, about 0.01 mg/kgto about 0.1 mg/kg, about 0.01 mg/kg to about 0.2 mg/kg, about 0.01mg/kg to about 0.5 mg/kg, about 0.01 mg/kg to about 1 mg/kg, about 0.02mg/kg to about 0.05 mg/kg, about 0.02 mg/kg to about 0.1 mg/kg, about0.02 mg/kg to about 0.2 mg/kg, about 0.02 mg/kg to about 0.5 mg/kg,about 0.02 mg/kg to about 1 mg/kg, about 0.05 mg/kg to about 0.1 mg/kg,about 0.05 mg/kg to about 0.2 mg/kg, about 0.05 mg/kg to about 0.5mg/kg, about 0.05 mg/kg to about 1 mg/kg, about 0.1 mg/kg to about 0.2mg/kg, about 0.1 mg/kg to about 0.5 mg/kg, about 0.1 mg/kg to about 1mg/kg, about 0.2 mg/kg to about 0.5 mg/kg, about 0.2 mg/kg to about 1mg/kg, or about 0.5 mg/kg to about 1 mg/kg. The therapeuticallyeffective amount can be about 0.001 mg/kg, about 0.002 mg/kg, about0.005 mg/kg, about 0.01 mg/kg, about 0.02 mg/kg, about 0.05 mg/kg, about0.1 mg/kg, about 0.2 mg/kg, about 0.5 mg/kg, or about 1 mg/kg. Thetherapeutically effective amount can be at least about 0.001 mg/kg,about 0.002 mg/kg, about 0.005 mg/kg, about 0.01 mg/kg, about 0.02mg/kg, about 0.05 mg/kg, about 0.1 mg/kg, about 0.2 mg/kg, or about 0.5mg/kg. The therapeutically effective amount can be at most about 0.002mg/kg, about 0.005 mg/kg, about 0.01 mg/kg, about 0.02 mg/kg, about 0.05mg/kg, about 0.1 mg/kg, about 0.2 mg/kg, about 0.5 mg/kg, or about 1mg/kg. The therapeutically effective amount can be about 0.1 mg/kg toabout 50 mg/kg. The therapeutically effective amount can be about 0.1mg/kg to about 0.2 mg/kg, about 0.1 mg/kg to about 0.5 mg/kg, about 0.1mg/kg to about 1 mg/kg, about 0.1 mg/kg to about 2 mg/kg, about 0.1mg/kg to about 5 mg/kg, about 0.1 mg/kg to about 10 mg/kg, about 0.1mg/kg to about 20 mg/kg, about 0.1 mg/kg to about 50 mg/kg, about 0.2mg/kg to about 0.5 mg/kg, about 0.2 mg/kg to about 1 mg/kg, about 0.2mg/kg to about 2 mg/kg, about 0.2 mg/kg to about 5 mg/kg, about 0.2mg/kg to about 10 mg/kg, about 0.2 mg/kg to about 20 mg/kg, about 0.2mg/kg to about 50 mg/kg, about 0.5 mg/kg to about 1 mg/kg, about 0.5mg/kg to about 2 mg/kg, about 0.5 mg/kg to about 5 mg/kg, about 0.5mg/kg to about 10 mg/kg, about 0.5 mg/kg to about 20 mg/kg, about 0.5mg/kg to about 50 mg/kg, about 1 mg/kg to about 2 mg/kg, about 1 mg/kgto about 5 mg/kg, about 1 mg/kg to about 10 mg/kg, about 1 mg/kg toabout 20 mg/kg, about 1 mg/kg to about 50 mg/kg, about 2 mg/kg to about5 mg/kg, about 2 mg/kg to about 10 mg/kg, about 2 mg/kg to about 20mg/kg, about 2 mg/kg to about 50 mg/kg, about 5 mg/kg to about 10 mg/kg,about 5 mg/kg to about 20 mg/kg, about 5 mg/kg to about 50 mg/kg, about10 mg/kg to about 20 mg/kg, about 10 mg/kg to about 50 mg/kg, or about20 mg/kg to about 50 mg/kg. The therapeutically effective amount can beabout 0.1 mg/kg, about 0.2 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about2 mg/kg, about 5 mg/kg, about 10 mg/kg, about 20 mg/kg, or about 50mg/kg. The therapeutically effective amount can be at least about 0.1mg/kg, about 0.2 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 2 mg/kg,about 5 mg/kg, about 10 mg/kg, or about 20 mg/kg. The therapeuticallyeffective amount can be at most about 0.2 mg/kg, about 0.5 mg/kg, about1 mg/kg, about 2 mg/kg, about 5 mg/kg, about 10 mg/kg, about 20 mg/kg,or about 50 mg/kg.

The individual treated can be a mammal. The mammal can be a mouse, rat,rabbit, dog, cat, horse, cow, sheep, pig, goat, llama, alpaca, or yak.The individual can be a dog, cat, or a horse. The individual to betreated can be a human.

Methods of Production

The polypeptide comprising an FGF17, IGF, or BMP7 ligand amino acidsequence can be purified or synthesized in any suitable manner. Anucleic acid encoding the polypeptide can be cloned into a suitablevector and expressed in a suitable cellular system. The cellular systemcan be a prokaryotic cell system. The cellular system can be aeukaryotic cell system. The cellular system can be a mammalian cellsystem. The polypeptide may be expressed from Eschericia coli. Thepolypeptide may be expressed from a yeast cell, including withoutlimitations, Saccharomyces cerevisiae, Pichia pastoris, Kluyveromyceslactis, Hansenula polymorpha, or Yarrowia lipolytica. The polypeptidemay be expressed from a mouse myeloma cell, including withoutlimitations, NS0, Sp2/0, and FO. The polypeptide may be expressed from achinese hamster ovary (CHO) cell. The polypeptide may be expressed by amammalian cell, including without limitations, a COS cell, a Vero cell,or a BHK cell. The polypeptide may be expressed from a human cell,including without limitations a HeLa cell, a HEK-293 cell, a CAP cell, aCAP-T cell, a PER.C6® cell.

The supernatants from such an expression system can be subjected to oneor more purification steps involving centrifugation,ultracentrifugation, filtration, diafiltration, tangential-flowfiltration, dialysis, chromatography (e.g., cation exchange, ionexchange, hydrophobic interaction, reverse phase, affinity, or sizeexclusion). The polypeptides can be purified to an extent suitable forhuman administration. Additionally, polypeptides can be synthesized forinclusion in a formulation to be administered to a human individual. Thepolypeptides can be produced by a suitable peptide synthesis method,such as solid-phase synthesis.

The mammalian expression vector pmax Cloning can be used to makeC-terminally 6×His-tagged, StrepII-tagged, and human IgG1 Fc-taggedvectors. The DNA fragments encoding the secreted myogenic factors areamplified by PCR from human open reading frame (ORF) clones, andsubsequently inserted into the tagged vectors by In-Fusion cloningtechnology (Takara Bio Inc.). The expression vectors carrying thesecreted myogenic factors are transiently transfected into ExpiCHO-Scells at a density of 6×10⁶ per ml by using ExpiFectamine CHOtransfection kit (Thermo Scientific).

The expressed myogenic factors with different tags in the culturesupernatants are affinity-purified by using different purificationmedia. The polypeptide can comprise an Fc region. For these polypeptidesa matrix or resin comprising Protein A, Protein G, protein L or anycombination thereof can be used. The matrix or resin may suitably beloaded onto a column for ease in batch purification.

Purification of Immunoglobulin Fusion Proteins

The heterologous sequence may comprise an immunoglobulin or a fragmentthereof. When the polypeptide comprises an immunoglobulin or a fragmentthereof, the polypeptide may be purified by means of protein A, G, or Laffinity. Protein A and G are cell surface proteins found inStaphylococcus aureus. They have the property of binding the Fc regionof a mammalian antibody, in particular of IgG class antibodies. For usein protein A or G affinity chromatography, protein A or G is coupled toa solid matrix such as crosslinked, uncharged agarose (Sepharose, freedfrom charged fraction of natural agarose), trisacryl, crosslinkeddextran or silica-based materials. Methods for such are commonly knownin the art, e.g. coupling via primary amino functions of the protein toa CNBr-activated matrix. Protein A binds with high affinity and highspecificity to the Fc portion of IgG, that is the Cγ2-Cγ3 interfaceregion of IgG as described in Langone et al., 1982, supra. Inparticular, it binds strongly to the human allotypes or subclasses IgG1,IgG2, IgG3 and the mouse allotypes or subclasses IgG2a, IgG2b, IgG3.

After purification by Protein A, G, or L the bound fraction can beeluted and passed over or through an additional resin or matrixcomprising one or more ion exchange columns. The first ion exchanger isgenerally an anion exchanger resin. The pH of buffer used for loadingand running the first ion exchanger is set as to put opposing totalchange on the Fc comprising fusion polypeptide and the protein A to beseparated by means of the ion exchanger in a flow-through mode accordingto the present invention, taking the pI's of the Fc comprising fusionpolypeptide and protein A into account. The mode of operation of a firstanion exchanger according to the present invention requires bufferexchange of the acidic or neutralized eluate from the protein A affinitychromatography step with the equilibrium buffer of the first anionexchanger. After the first anion exchanger, the Fc comprising fusionpolypeptide is ready for use in applications or may be deemed to requirefurther polishing by customary purification methods. In a furtherpreferred embodiment, the first ion exchange step is followed by asecond ion exchange step in which second step the antibody is loaded andbound by the second ion exchange medium and is eluted with a bufferother than the loading buffer, by means of increased salt and/or pH, asan essentially monomeric, non-aggregated antibody.

In the methods disclosed herein at least 70%, 80%, or 90% of the Fccomprising fusion polypeptide loaded onto the first ion exchanger can berecovered in the flow-through of the ion-exchanger.

Master Cell Bank and Transgenic Cells

Described herein are master cell banks that can comprise a cell thatcomprises a nucleic acid encoding one or more IGF ligand or IGF2 fusionpolypeptides integrated into its genome creating a transgenic cell-line.The master cell bank can comprise a plurality of cells that eachcomprise a nucleic acid encoding an IGF ligand or IGF2 fusionpolypeptide. The nucleic acid can be maintained extrachromosomally on aplasmid or yeast artificial chromosome. The nucleic acid can beintegrated into a chromosomal location. The cell can be a yeast cell.The yeast can be Pichia pastoris or Saccharomyces cerevisiae. The cellcan be a mammalian cell. The mammalian cell can be a 293T cell orderivative thereof (e.g., 293T-Rex). The cell can be a bacterial cell.

The transgenic mammalian, yeast, or bacterial cell can be a master cellbank that comprises a cryopreservative suitable for freezing to at leastabout −80° or below. The master cell bank can comprise glycerol or DMSOat between about 10 and about 30%, and can be suitable for long-termstorage at about −80° or below. The master cell bank can preserve atransgenic mammalian, yeast, or bacterial strain for at least 1, 2, 3,4, 5, 6, 7, 8, 9, 10, or more years.

Pharmaceutically Acceptable Excipients. Carriers, and Diluents

The polypeptides comprising an FGF17, IGF2, or BMP7 amino acid sequenceand an amino acid sequence from a heterologous polypeptide orcombinations of an Fibroblast Growth Factor Receptor agonist and aglycosaminoglycan, an Insulin-like Growth Factor 1 Receptor (IGF1R)agonist and a short chain fatty acid, and BMP receptor agonists and mTORactivators and/or glycosaminoglycans described herein can beadministered in a pharmaceutical composition that comprises one or morepharmaceutically acceptable excipients, carriers, or diluents. The exactcomponents can differ based upon the preferred route of administration.The excipients used in a pharmaceutical composition can provideadditional function to the polypeptide by making the polypeptidesuitable for a particular route of administration (e.g., intravenous,topical, subcutaneous, or intramuscular), increasing polypeptidestability, increasing penetration of a desired tissue (e.g., muscle orskin), increasing residence time at particular site, increasingsolubility, enhancing the efficacy of the polypeptide, and/or reducinginflammatory reactions coincident with administration.

The compositions can be included in a pharmaceutical composition with asolubilizing emulsifying, or dispersing agent. The solubilizing agentcan allow high-concentration solutions of fusion polypeptides thatexceed at least about 2 mg/mL, 5 mg/mL, 10 mg/mL, 15 mg/mL, or 20 mg/mL.Carbomers in an aqueous pharmaceutical composition serve as emulsifyingagents and viscosity modifying agents. The pharmaceutically acceptableexcipient can comprise or consist of a carbomer. The carbomer cancomprise or consist of carbomer 910, carbomer 934, carbomer 934P,carbomer 940, carbomer 941, carbomer 1342, or combinations thereof.Cyclodextrins in an aqueous pharmaceutical composition serve assolubilizing and stabilizing agents. The pharmaceutically acceptableexcipient can comprise or consist of a cyclodextrin. The cyclodextrincan comprise or consist of alpha cyclodextrin, beta cyclodextrin, gammacyclodextrin, or combinations thereof. Lecithin in a pharmaceuticalcomposition may serve as a solubilizing agent. The solubilizing agentcan comprise or consist of lecithin. Poloxamers in a pharmaceuticalcomposition serve as emulsifying agents, solubilizing agents, anddispersing agents. The pharmaceutically acceptable excipient cancomprise or consist of a poloxamer. The poloxamer can comprise orconsist of poloxamer 124, poloxamer 188, poloxamer 237, poloxamer 338,poloxamer 407, or combinations thereof. Polyoxyethylene sorbitan fattyacid esters in a pharmaceutical composition serve as emulsifying agents,solubilizing agents, surfactants, and dispersing agents. Thepharmaceutically acceptable excipient can comprise or consist of apolyoxyethylene sorbitan fatty acid ester. The polyoxyethylene sorbitanfatty acid ester can comprise or consist of polysorbate 20, polysorbate21, polysorbate 40, polysorbate 60, polysorbate 61, polysorbate 65,polysorbate 80, polysorbate 81, polysorbate 85, polysorbate 120, orcombinations thereof. Polyoxyethylene stearates in a pharmaceuticalcomposition serve as emulsifying agents, solubilizing agents,surfactants, and dispersing agents. The pharmaceutically acceptableexcipient can comprise or consist of a polyoxyethylene stearate. Thepolyoxyethylene stearate can comprise or consist of polyoxyl 2 stearate,polyoxyl 4 stearate, polyoxyl 6 stearate, polyoxyl 8 stearate, polyoxyl12 stearate, polyoxyl 20 stearate, polyoxyl 30 stearate, polyoxyl 40stearate, polyoxyl 50 stearate, polyoxyl 100 stearate, polyoxyl 150stearate, polyoxyl 4 distearate, polyoxyl 8 distearate, polyoxyl 12distearate, polyoxyl 32 distearate, polyoxyl 150 distearate, orcombinations thereof. Sorbitan esters in a pharmaceutical compositionserve as emulsifying agents, solubilizing agents, and non-ionicsurfactants, and dispersing agents. The pharmaceutically acceptableexcipient can comprise or consist of a sorbitan ester. The sorbitanester can comprise or consist of sorbitan laurate, sorbitan oleate,sorbitan palmitate, sorbitan stearate, sorbitan trioleate, sorbitansesquioleate, or combinations thereof. Solubility can be achieved with aprotein carrier. The protein carrier can comprise recombinant humanalbumin.

The polypeptides comprising an FGF17, IGF2, or BMP7 amino acid sequenceand an amino acid sequence from a heterologous polypeptide orcombinations of an Fibroblast Growth Factor Receptor agonist and aglycosaminoglycan, an Insulin-like Growth Factor 1 Receptor (IGF1R)agonist and a short chain fatty acid, and BMP receptor agonists and mTORactivators and/or glycosaminoglycans described herein can be formulatedto increase stability. Polypeptides in aqueous formulations may requirestabilization to prevent degradation. The stabilizer can comprise pHbuffers, salts, amino acids, polyols/disaccharides/polysaccharides,liposomes, surfactants, antioxidants, reducing agents, or chelatingagents. The stabilizer can comprise or consist of a polyol/non-reducingsugar. The non-reducing sugar can comprise or consist of sucrose,mannitol, trehalose, raffinose, stachyose, xylitol, starch, verbascose,or combinations thereof. Polypeptides can be encapsulated in liposomesto increase stability. The stabilizer can comprise or consist ofliposomes. The liposomes can comprise or consist ofipalmitoylphosphatidylcholine (DPPC) liposomes,phosphatidylcholine:cholesterol (PC:Chol) (70:30) liposomes, ordipalmitoylphosphatidylcholine: dipalmitoylphosphatidylserine(DPPC:DPPS) liposomes (70:30). Non-ionic surfactants can increase thestability of a polypeptide. The stabilizer can comprise or consist of anon-ionic surfactant. The non-ionic surfactant can comprise or consistof polysorbates (e.g., poly sorbate 80, poly sorbate 20),alkylsaccharides alkyl ethers and alkyl glyceryl ethers, polyoxyethelene(4) lauryl ether; polyoxyethylene cetyl ethers, polyoxyethylene stearylethers, sorbitan fatty acid esters, polyoxyethylene fatty acid esters,or combinations thereof. The polypeptide can be formulated with aprotein surfactant, such as recombinant human serum albumin as astabilizer. Antioxidants or reducing agents can increase the stabilityof a polypeptide. The stabilizer can comprise or consist of anantioxidant or reducing agent. The reducing agent can comprise orconsist of dithiothreitol, ethylenediaminetetraacetic acid,2-Mercaptoethanol, Tris(2-carboxyethyl)phosphine hydrochloride,Tris(hydroxypropyl)phosphine, or combinations thereof. The antioxidantcan comprise or consist of methionine, ascorbic acid, citric acid, alphatocopherol, sodium bisulfite, ascorbyl palmitate, erythorbic acid, orcombinations thereof. Chelating agents can stabilize polypeptides byreducing the activity of proteases. The stabilizer can comprise orconsist of a chelating agent. The chelating agent can comprise orconsist of ethylenediaminetetraacetic acid (EDTA), ethyleneglycol-bis(3-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), metalcomplexes (e.g. Zn-protein complexes), or combinations thereof. Bufferagents can stabilize polypeptides by reducing the acid hydrolysis ofpolypeptides. The stabilizer can comprise or consist of a buffer agent.The buffer agent can comprise or consist of sucrose octa-sulfate,ammonium carbonate, ammonium phosphate, boric acid, sodium citrate,potassium citrate, lactic acid, 3-(N-morpholino)propanesulfonic acid(MOPS), 2-(N-morpholino)ethanesulfonic acid (MES),hydroxymethylaminomethane (Tris), calcium carbonate, calcium phosphateor combinations thereof.

The polypeptides comprising an FGF17, IGF2, or BMP7 amino acid sequenceand an amino acid sequence from a heterologous polypeptide orcombinations of an Fibroblast Growth Factor Receptor agonist and aglycosaminoglycan, an Insulin-like Growth Factor 1 Receptor (IGF1R)agonist and a short chain fatty acid, and BMP receptor agonists and mTORactivators and/or glycosaminoglycans described herein also may beentrapped in or associated with microcapsules prepared, for example, bycoacervation techniques or by interfacial polymerization (for example,hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively), in colloidaldelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules), or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences,16th edition, Oslo, A., Ed., (1980).

The polypeptides comprising an FGF17, IGF2, or BMP7 amino acid sequenceand an amino acid sequence from a heterologous polypeptide orcombinations of an Fibroblast Growth Factor Receptor agonist and aglycosaminoglycan, an Insulin-like Growth Factor 1 Receptor (IGF1R)agonist and a short chain fatty acid, and BMP receptor agonists and mTORactivators and/or glycosaminoglycans described herein may be formulatedor delivered with an anti-inflammatory agent. The anti-inflammatoryagent can comprise or consist of a corticosteroid. The corticosteroidcan comprise or consist of hydrocortisone, cortisone, ethamethasoneb(Celestone), prednisone (Prednisone Intensol), prednisolone (Orapred,Prelone), triamcinolone (Aristospan Intra-Articular, AristospanIntralesional, Kenalog), methylprednisolone (Medrol, Depo-Medrol,Solu-Medrol), or dexamethasone (Dexamethasone Intensol). Theanti-inflammatory can comprise or consist of a non-steroidalanti-inflammatory (NSAID). The NSAID can comprise or consist of aspirin,celecoxib, diclofenac, diflunisal, etodolac, ibuprofen, indomethacin,ketoprofen, ketorolac, nabumetone, naproxen, oxaprozin, piroxicam,salsalate, sulindac, or tolmetin.

The polypeptides comprising an FGF17, IGF2, or BMP7 amino acid sequenceand an amino acid sequence from a heterologous polypeptide orcombinations of an Fibroblast Growth Factor Receptor agonist and aglycosaminoglycan, an Insulin-like Growth Factor 1 Receptor (IGF1R)agonist and a short chain fatty acid, and BMP receptor agonists and mTORactivators and/or glycosaminoglycans described herein can be included ina pharmaceutical composition suitable for intravenous administrationcomprising one or more pharmaceutically acceptable excipients, carriers,and diluents. The polypeptides of the current disclosure can beadministered suspended in a sterile solution. The solution can be onecommonly used for administration of biological formulations, andcomprises, for example, about 0.9% NaCl or about 5% dextrose. Thesolution can further comprise one or more of: buffers, for example,acetate, citrate, histidine, succinate, phosphate, potassium phosphate,bicarbonate and hydroxymethylaminomethane (Tris); surfactants, forexample, polysorbate 80 (Tween 80), polysorbate 20 (Tween 20), andpoloxamer 188; polyol/disaccharide/polysaccharides, for example,glucose, dextrose, mannose, mannitol, sorbitol, sucrose, trehalose, anddextran 40; amino acids, for example, glycine, histidine, leucine, orarginine; antioxidants, for example, ascorbic acid, methionine; orchelating agents, for example, EDTA, or EGTA.

The polypeptides comprising an FGF17, IGF2, or BMP7 amino acid sequenceand an amino acid sequence from a heterologous polypeptide orcombinations of an Fibroblast Growth Factor Receptor agonist and aglycosaminoglycan, an Insulin-like Growth Factor 1 Receptor (IGF1R)agonist and a short chain fatty acid, and BMP receptor agonists and mTORactivators and/or glycosaminoglycans described herein can be included ina pharmaceutical composition suitable for intramuscular or subcutaneousadministration comprising one or more pharmaceutically acceptableexcipients, carriers, and diluents. Formulations suitable forintramuscular or subcutaneous injection can include physiologicallyacceptable sterile aqueous or non-aqueous solutions, dispersions,suspensions or emulsions, and sterile powders for reconstitution intosterile injectable solutions or dispersions. Examples of suitableaqueous and non-aqueous carriers, diluents, solvents, or vehiclesinclude ethanol, polyols (inositol, propyleneglycol,polyethylene-glycol, glycerol, cremophor and the like) and suitablemixtures thereof, vegetable oils (such as olive oil) and injectableorganic esters such as ethyl oleate. Proper fluidity is maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersions, and by the use ofsurfactants. Formulations suitable for subcutaneous injection alsocontain optional additives such as preserving, wetting, emulsifying, anddispensing agents.

The polypeptides comprising an FGF17, IGF2, or BMP7 amino acid sequenceand an amino acid sequence from a heterologous polypeptide orcombinations of an Fibroblast Growth Factor Receptor agonist and aglycosaminoglycan, an Insulin-like Growth Factor 1 Receptor (IGF1R)agonist and a short chain fatty acid, and BMP receptor agonists and mTORactivators and/or glycosaminoglycans described herein can be formulatedfor topical administration as a cream, gel, paste, ointment, oremulsion. Excipients in a cream, gel, paste, ointment, or emulsion cancomprise gelatin, casein, lecithin, gum acacia, cholesterol, tragacanth,stearic acid, benzalkonium chloride, calcium stearate, glycerylmonostearate, cetostearyl alcohol, cetomacrogol emulsifying wax,sorbitan esters, polyoxyethylene alkyl ethers, polyoxyethylene castoroil derivatives, polyoxyethylene sorbitan fatty acid esters,polyethylene glycols, polyoxyethylene stearates, colloidol silicondioxide, phosphates, sodium dodecyl sulfate, carboxymethylcellulosecalcium, carboxymethylcellulose sodium, methylcellulose,hydroxyethylcellulose, hydroxypropylcellulose,hydroxypropylmethycellulose phthalate, noncrystalline cellulose,magnesium aluminum silicate, triethanolamine, polyvinyl alcohol,polyvinylpyrrolidone, sugars, and starches.

The excipient used with the polypeptides comprising an FGF17, IGF2, orBMP7 amino acid sequence and an amino acid sequence from a heterologouspolypeptide or combinations of an Fibroblast Growth Factor Receptoragonist and a glycosaminoglycan, an Insulin-like Growth Factor 1Receptor (IGF1R) agonist and a short chain fatty acid, and BMP receptoragonists and mTOR activators and/or glycosaminoglycans described hereinwill allow for storage, formulation, or administration of highlyconcentrated formulations. In certain embodiments, a highly concentratedfusion polypeptide(s) comprises at least about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 25, 20, 25, 40, 45, 50 or more milligrams per milliliter.

The polypeptides and/or compositions of the current disclosure can beshipped/stored lyophilized and reconstituted before administration.Lyophilized ligand fusion polypeptide formulations can comprise abulking agent such as, mannitol, sorbitol, sucrose, trehalose, anddextran 40. The lyophilized formulation can be contained in a vialcomprised of glass. The fusion polypeptides when formulated, whetherreconstituted or not, can be buffered at a certain pH, generally lessthan 7.0. In certain embodiments, the pH can be between 4.5 and 6.5, 4.5and 6.0, 4.5 and 5.5, 4.5 and 5.0, or 5.0 and 6.0.

Kits

Also described herein are kits comprising one or more of thepolypeptides comprising an FGF17, IGF2, or BMP7 amino acid sequence andan amino acid sequence from a heterologous polypeptide or combinationsof an Fibroblast Growth Factor Receptor agonist and a glycosaminoglycan,an Insulin-like Growth Factor 1 Receptor (IGF1R) agonist and a shortchain fatty acid, and BMP receptor agonists and mTOR activators and/orglycosaminoglycans described herein in a suitable container and one ormore additional components selected from: instructions for use; adiluent, an excipient, a carrier, and a device for administration.

In an aspect, described herein is a method of preparing a soft tissue ormuscle disease or disorder treatment comprising admixing one or morepharmaceutically acceptable excipients, carriers, or diluents andpolypeptides comprising an FGF17, IGF2, or BMP7 amino acid sequence andan amino acid sequence from a heterologous polypeptide or combinationsof an Fibroblast Growth Factor Receptor agonist and a glycosaminoglycan,an Insulin-like Growth Factor 1 Receptor (IGF1R) agonist and a shortchain fatty acid, and BMP receptor agonists and mTOR activators and/orglycosaminoglycans described herein. In an aspect, described herein is amethod of preparing a soft tissue or muscle disease or disordertreatment for storage or shipping comprising lyophilizing one or moreantibodies of the current disclosure.

The inventions disclosed herein will be better understood from theexperimental details which follow. However, one skilled in the art willreadily appreciate that the specific methods and results discussed aremerely illustrative of the inventions as described more fully in theclaims which follow thereafter. Unless otherwise indicated, thedisclosure is not limited to specific procedures, materials, or thelike, as such may vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular embodiments onlyand is not intended to be limiting.

EXAMPLES Example 1—Expression and Purification of Recombinant Proteins

Mammalian expression plasmids carrying genes with different tags weretransiently transfected into CHO cells. The genes were expressed toproduce proteins that were subsequently secreted into the culturemedium. The proteins in the culture medium were visualized onpolyacrylamide gels and their activities were measured by in vitrofunctional assays. Then the recombinant proteins in the culture mediumwere affinity purified. The purified proteins were visualized onpolyacrylamide gels to evaluate the purity and assayed by in vitrofunctional assays to determine their biological activities.

Expression vector engineering: Mammalian expression vector pmax Cloningwas used to make C-terminally 6×His-tagged, StrepII-tagged, and humanIgG1 and IgG4 Fc-tagged vectors. The DNA fragments encoding the secretedmyogenic factors were amplified by PCR from human open reading frame(ORF) clones, and subsequently inserted into the tagged vectors byIn-Fusion cloning technology (Takara Bio Inc.).

Expressing secreted myogenic polypeptides: The expression vectorscarrying the secreted myogenic factors were transiently transfected intoExpiCHO-S cells at a density of 6×10⁶ per ml by using ExpiFectamine CHOtransfection kit (Thermo Scientific). After 18-22 hours, CHO feed andenhancer were added into the transfected culture. Then the expressedproteins were monitored by SDS-PAGE every 24 hours to achieve maximalexpression level. In most of the cases, cell culture was collected atday 4, and cells were spun down. The supernatant was spun down again toget rid of cellular debris. The clarified culture supernatant containingthe secreted myogenic factors was stored at −80° C. or immediatelyprocessed for use.

Measuring expression level of secreted myogenic polypeptides: To measurethe improved expression level of the secreted myogenic factors, threeprotein analytical techniques were applied: sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), Western blotting, andenzyme-linked immunosorbent assay (ELISA). Western Blots were performedto identify the myogenic factors. ELISAs were used to measure theabsolute amount of myogenic factors in the culture supernatant.

Isolation of engineered myogenic polypeptides: The expressed myogenicfactors with different tags in the culture supernatants wereaffinity-purified by using different purification media. For Fc-fusionfactors, either Protein A magnetic beads (GenScript) or Protein Amembrane column (Takara Bio Inc.) were used to specifically bind to theFc-fusion factors. For 6×His-tagged factors, NTA-magnetics beads (NEB)were used to isolate the factor.

Example 2—Purified IGF2-hFcm Promoted Differentiation of Human MyoblastCells

FIG. 1A: The suspension CHO cells were transiently transfected with theIGF2-hFcm encoding plasmid. IGF2-hFcm was affinity-purified by Protein Amembrane column. The purified IGF2-hFcm was added into the culture ofhuman myoblast cells for 96 hours. Myosin heavy chain (MyHC) wasimmunostained and imaged by a fluorescence microscope. The percentagearea of MyHC of human myoblasts treated with the purified IGF2-hFcm issignificantly higher than the percentage area of MyHC of human myoblaststreated with the vehicle control (One-Way ANOVA Tukey Honest SignificantDifference, n=2-6).

Condition % MyHC SD p-value Vehicle control 1.787 0.186  33 nM IGF2-hFcm3.734 0.790 0.012  66 nM IGF2-hFcm 5.922 0.795 3.20E−05 133 nM IGF2-hFcm7.568 0.538 1.46E−06

Example 3 IGF2-LhFc4 Promoted Differentiation of Human Myoblast Cells

The suspension CHO cells were transiently transfected with theIGF2-LhFc4 encoding plasmid. IGF2-LhFc4 was affinity-purified by ProteinA membrane column. The purified IGF2-LhFc4 was added into the culture ofhuman myoblast cells for 96 hours with daily media change. Myosin heavychain (MyHC) was immunostained and imaged by a fluorescence microscope.The percentage area of MyHC of human myoblasts treated with the purifiedIGF2-LhFc4 is significantly higher than the percentage area of MyHC ofhuman myoblasts treated with the vehicle control (One-Way ANOVA TukeyHonest Significant Difference, n=2-6).

Condition % MyHC SD p-value Vehicle control 1.384 0.285 hFc4L-IGF2 5.8200.319 0.011 IGF2-hFc4 6.901 0.537 0.004 IGF2-LhFc4 6.237 1.848 0.007

Example 4—Purified HSA-L-IGF2R61A Differentiation of Human MyoblastCells

The suspension CHO cells were transiently transfected with theHSA-L-IGF2R61A encoding plasmid. HSA-L-IGF2R61A was affinity-purified byProtein A membrane column. The purified HSA-L-IGF2R61A was added intothe culture of human myoblast cells for 96 hours with daily mediachange. Myosin heavy chain (MyHC) was immunostained and imaged by afluorescence microscope. The percentage area of MyHC of human myoblaststreated with the purified HSA-L-IGF2R61A is significantly higher thanthe percentage area of MyHC of human myoblasts treated with the vehiclecontrol (One-Way ANOVA Tukey Honest Significant Difference, n=2-6).

Condition % MyHC SD p-value Vehicle control 0.34 0.221 HSA-IGF2 7.0792.009 0.013 HSA-IGF2R61A 6.914 2.691 0.014

Example 5 IGF2 and IGF2 Receptors are Expressed in Human Myoblast

Bar graph and quantitation table of IGF2 and IGF2 receptor RNASeqexpression in young (17-21 year old caucasian males) and aged humanmyoblast (68-69 year old caucasian males) cell lines. Myoblast werecultured growth media (GM) or 96h in fusion media (FM). Fresh media wasadded every 24h. Mean±SEM. n=6. Expression are expressed as FPKM.Significant p-values (Young GM˜Aged GM: 3.54E-04).

n = 6 IGF2 (FPKM) SEM p-val (n = 6) Young GM 13.11 3.275 — Aged GM 3.4131.12 3.54E−04 Young FM 17.68 6.42 — Aged FM 13.08 3.67 n.s.

n = 6 IGF2R (FPKM) SEM P-val (n = 6) Young GM 74.93 9.45 — Aged GM 75.016.89 n.s. Young FM 82.35 3.43 — Aged FM 88.44 9.86 n.s.

Example 6 Sodium Butyrate Enhances Muscle Fusion

Mouse myoblasts were treated with PBS or sodium butyrate atconcentrations 0.1 nM, 1 nM, and 10 nM. Myoblasts were cultured for 48hours, with fresh media added every 24 hours. Cells were pulsed for 2-5hours with EdU (30 uM), ethanol fixed, stained with Hoescht 3342,immunostained for proliferation—as measured by the percent of cellsstaining positive for EdU (% EdU)—, and immunostained fordifferentiation—as measured by the increase in cellular area stainingpositive for embryonic myosin heavy chain (% eMyHC) relative to thenegative controls, which received media and vehicle only. When comparedto untreated myoblasts, the cells treated with 1 nM of sodium butyratehad increased rates of fusion, as depicted in FIG. 2A. Significance wasdetermined by a p-value less than 0.05 by the one-way ANOVA Tukey HonestSignificant Difference test.

FIG. 2A: Bar graph of fusion index in response to sodium butyrate(NaBut) compared to vehicle. Myoblast were cultured 48h in the presenceof NaBut at indicated dose. Fresh media and NaBut were added every 24h.Mean∓S.D. Table quantitation of fusion index and p-values also shown.(*p<0.05 by Student's Two-tailed T-test, n=3-5)

Condition Fusion Index (nuclei/myotube) p-value vehicle 7.56 — NaBut 0.1nM 8.44 n.s. NaBut 1 nM 31.60 2.02E−3 NaBut 10 nM 14.00 n.s.

Example 7 Sodium Butyrate Enhances IGF2 Activity

Human myoblast cells were treated with either PBS (vehicle), IGF2 (15ng/mL), sodium butyrate, or IGF2 and sodium butyrate. Fresh media wasadded every 24 hours. After 96 hours, cells were pulsed for 2-5 hourswith EdU (30 uM), ethanol fixed, stained with Hoescht 3342,immunostained for proliferation—as measured by the percent of cellsstaining positive for EdU (% EdU)—, and immunostained fordifferentiation—as measured by the increase in cellular area stainingpositive for embryonic myosin heavy chain (% eMyHC) relative to thenegative controls, which received media and vehicle only. The total areaof eMyHc positive cells was analyzed, and treated cells were compared tocells treated with the vehicle alone. Cells that had been treated withIGF alone and two conditions in which cells had been treated with IGF2and sodium butyrate produced a significant increase in the amount ofdifferentiation. There was a significant increase in the total area ofeMyHC cells in the cells treated with 1 nM and 100 nM of sodium butyrateand IGF2, compared to the cells treated with IGF2 alone. Significancewas determined by a p-value less than 0.05 by the one-way ANOVA TukeyHonest Significant Difference test.

FIG. 2B: Bar graph of fusion index of mouse myoblast in response tosodium butyrate (NaBut) compared to vehicle. Mouse myoblast werecultured 48h in the presence of NaBut at indicated dose. Fresh media andNaBut were added every 24h. Mean∓S.D. Table quantitation of fusion indexand p-values shown. (*p<0.05 by Student's Two-tailed T-test, n=3-5).Significant p-values (Vehicle˜IGF2: 0.015 ug/mL: 6.33E-06,Vehicle˜NaBut: 1 nM IGF2: 0.015 ug/mL: 1.79E-11, Vehicle˜NaBut: 100 nMIGF2: 0.015 ug/mL: 1.79E-11)

Table of data for FIG. 2B Condition % eMyHC SD p-value Vehicle 6.8131.695 IGF2: 0.015 ug/mL 10.843 1.308 NaBut: 1 nM 2.321 0.374 NaBut: 10nM 6.199 1.174 NaBut: 100 nM 8.341 0.477 NaBut: 1 nM IGF2: 0.015 ug/mL28.387 1.036 1.79E−11 NaBut: 10 nM IGF2: 0.015 ug/mL 9.274 0.654 NaBut:100 nM IGF2: 0.015 ug/mL 29.239 3.185 1.79E−11

FIG. 2C Bar graph quantitation of % Area eMyHC+ human myoblast inresponse to indicated treatment compared to IGF2 (15 ng/mL). Myoblastwere cultured 96h in the presence of BMP7 at indicated dose. Fresh mediaand BMP7 was added every 24h. Mean∓S.D. (*p<0.05 by One-Way Anova TukeyHonest Significant Difference, n=2-12)

Table of data for FIG. 2C Condition % eMyHC SD p-value IGF2: 0.015 ug/mL10.843 1.308 NaBut: 1 nM IGF2: 0.015 ug/mL 28.387 1.036 6.18E−8 NaBut:10 nM IGF2: 0.015 ug/mL 9.274 0.654 NaBut: 100 nM IGF2: 0.015 ug/mL29.239 3.185 3.50E−3

Example 8 Sodium Butyrate Enhances IGF2 Activity

Human myoblast cells were treated with either PBS (vehicle), IGF2 (15ng/mL), sodium butyrate, or IGF2 and sodium butyrate. Fresh media wasadded every 24 hours. After 48 hours, cells were pulsed for 2-5 hourswith EdU (30 uM), ethanol fixed, stained with Hoescht 3342,immunostained for proliferation—as measured by the percent of cellsstaining positive for EdU (% EdU)—, and immunostained fordifferentiation—as measured by the increase in cellular area stainingpositive for embryonic myosin heavy chain (%/eMyHC) relative to thenegative controls, which received media and vehicle only. The total areaof eMyHc positive cells was analyzed, and treated cells were compared tocells treated with the vehicle alone, as seen in FIG. 3A. Myoblasts thathad been treated with either 0.03 ug/mL of IGF2 or with IGF2 incombination with sodium butyrate showed a significant increase in theeMyHC+ area when compared to cells cultured with the vehicle alone.

FIG. 3A: Bar graph of % Area eMyHC+ age human myoblast (68 year oldcaucasian male) in response to indicated treatment compared to Vehicle(vehicle). Myoblast were cultured 96h in the presence of factors atindicated dose. Fresh media and factors were added every 24h. Mean∓S.D.Significant p-values (Vehicle˜IGF2: 0.03 ug/mL: 1.42E-08, Vehicle˜NaBut:1 nM IGF2: 0.03 ug/mL: 1.79E-11, Vehicle˜NaBut: 10 nM IGF2: 0.03 ug/mL:1.80E-11, Vehicle˜NaBut: 100 nM IGF2: 0.03 ug/mL: 1.79E-11). FIG. 3B Bargraph quantitation of % Area eMyHC+ human myoblast (68 year oldcaucasian male) in response to indicated treatment compared to IGF2 (15ng/mL). Myoblast were cultured 96h in the presence of BMP7 at indicateddose. Mean∓S.D. Significant p-values (IGF2˜NaBut: 1 nM IGF2: 0.03 ug/mL:1.88E-3, Vehicle˜NaBut: 10 nM IGF2: 0.03 ug/mL: 4.80E-3, Vehicle˜NaBut:100 nM IGF2: 0.03 ug/mL: 1.87E-3) (*p<0.05 by One-Way ANOVA Tukey HonestSignificant Difference, n=2-12) Table of data for FIG. 3A

Condition % eMyHC SD p-value Vehicle 6.813 1.695 — IGF2: 0.03 ug/mL16.620 1.301 1.42E−08 NaBut: 1 nM 2.321 0.374 NaBut: 10 nM 6.199 1.174NaBut: 100 nM 8.341 0.477 NaBut: 1 nM IGF2: 0.03 ug/mL 24.615 0.2581.79E−11 NaBut: 10 nM IGF2: 0.03 ug/mL 22.821 0.234 1.80E−11 NaBut: 100nM IGF2: 0.03 ug/mL 28.427 3.136 1.79E−11

The myoblasts that had been treated with a combination of IGF2 andsodium butyrate were compared to the cells treated with IGF2 alone.There was a significant increase in all cells treated with thecombination compared to cells treated with IGF2 alone, as depicted inFIG. 3B and Table 12. Significance was determined by a p-value less than0.05 by the one-way ANOVA Tukey Honest Significant Difference test.

Table of data for FIG. 3B Condition % eMyHC SD p-value IGF2: 0.03 ug/mL16.620 1.301 NaBut: 1 nM IGF2: 0.03 ug/mL 24.615 0.258 1.88E−3 NaBut: 10nM IGF2: 0.03 ug/mL 22.821 0.234 4.80E−3 NaBut: 100 nM IGF2: 0.03 28.4273.136 1.87E−3 ug/mL

Example 9 IGF2 Enhances MYOG Expression in DM1 Human Myoblast Cells

Bar graph of myogenic gene expression fold change in DM1 human myoblastin response to indicated treatment compared to FM (vehicle). Myoblastswere cultured 48h in the presence of factors (BMP7 50 ng/mL, Butyrate100 nM, IGF2 200 ng/mL). Mean∓S.D. Significant p-values (FM˜IGF2:4.94E-04, FM˜IGF2_NaBut: 6.53E-03) (*p<0.01) Table of mean and p-valueof MYF5, MYOD1, and MYOG (n=3).

TABLE of data MYF5 MYOD1 MYOG p- p- p- Condition MYF5 value MYOD1 valueMYOG value FM 1.000 1.000 1.000 BMP7 0.709 n.s. 0.709 n.s. 0.361 n.s.NaBut 1.128 n.s. 1.095 n.s. 1.020 n.s. IGF2 0.730 n.s. 1.252 n.s. 2.9724.94E−04 IGF2 0.820 n.s. 1.500 n.s. 3.483 6.53E−03 NaBut

Example 10 IGF2 Receptor is Expressed on Chondrocyte and Osteocytes

FIG. 4A: Bar graph showing IGF2 receptors are expressed oncartilage-associated cells. Data is derived from Ramilowsky et al.,Nature 2015.

Table of data for FIG. 4A RNA Expression (TPM) Cell Type IGF2RPreadipocyte (Subcutaneous) 27.083 Chondrocyte 47.63 Osteocyte 83.96Tenocyte 23.12

Example 11 IGF2 Treatment Promotes Proliferation and Fusion in DM1 HumanMyoblast (32 Year Old Caucasian Female) Cells

Bar graph of % EdU+ human myoblast (32 year old caucasian female) and %area MyHC in response to IGF2. Myoblast were cultured 72h forproliferation and 96h for fusion in the presence of indicated factor.Mean∓S.D. Mean∓SD. Significant p-values (EdU: Vehicle˜IGF2: 6.8E-3, %eMyHC Area: Vehicle˜IGF2: 1.9E-4) (*p<0.05 by Students Two-TailedT-test, n=3-6).

Table of data n = 3-6 EdU FC s.d. p-val Vehicle 1.0 0.02 IGF2 2.18 0.326.8E−3

Table of data n = 3 % eMyHC are s.d. p-val Vehicle 0.45 0.02 IGF2 5.490.54 1.9E−4

Example 12 IGF2 Enhances MYH3, CKM, and ATP1B1 Expression in DM1 HumanMyoblast (32 Year Old Caucasian Female) Cells

Bar graph of MYH3 and CKM expression fold change in DM1 human myoblast(32 year old caucasian female) in response to indicated treatmentcompared to vehicle. Myoblasts were cultured 96h in the presence offactors (IGF2 200 ng/mL). Mean∓S.D. Significant p-values (MYH3:Vehicle˜IGF2: 1.13E-03, CKM: Vehicle˜IGF2: 7.67E-03) Bar graph of ATP1B1expression fold change in DM1 human myoblast (32 year old caucasianfemale) in response to indicated treatment compared to FM (vehicle).Myoblasts were cultured 48h in the presence of factors (IGF2 200 ng/mL).Mean∓S.D. Significant p-values (Vehicle˜IGF2: 3.11E-05) (*p<0.05 byStudents Two-Tailed T-test, n=3).

Table of data MYH3 p-val CKM p-val Vehicle 1 1 IGF2 14.833 1.13E−035.165 7.67E−03

Table of data n = 3 ATP1B1 p-val Vehicle 1 IGF2 3.01789 3.11E−05

Example 13 Systemic Administration of IGF2/NaB Protects Against AgingInduced Muscle Dysfunction

Subcutaneous injection of IGF2 (50 ug/kg) or NaB (1.2 g/kg), IGF2/NaB(150 ug/kg; 1.2 g/kg) or vehicle (PBS) were administered to 21-24M oldmice for 14 days. Muscle function was assessed at days 13 and 14. Gripstrength force assessed at day 13. The first graph shows Bothlimb gripstrength force, **** p<0.0001, **p=0.0043, **p=0.001 (One-way ANOVA,multiple comparisons). Forelimb force, ****p<0.0001, *p=0.0368,*p=0.0187 (One-way ANOVA, multiple comparisons). Treadmill performancemeasured at day 14 using an induced treadmill running model set toprogressively increase speed 2 m/min every subsequent 2 min. Distanceran shown. ***p=0.0005, *p=0.0459, ****p<0.0001 (One-way ANOVA, multiplecomparisons) Time to exhaustion ***p=0.0002, **p=0.0024 (One-way ANOVA,multiple comparisons) Maximum speed ***p=0.0004, **p=0.0013 Work in kj**p=0.0026, **p=0.0035 (One-way ANOVA, multiple comparisons).

Example 14 Systemic Administration of IGF2/NaB is Safe

Subcutaneous injection of vehicle or IGF2/NaB were administered to 21Mold mice for 14 days, blood and serum were collected to assess completeblood count and a metabolic panel for liver, kidney and pancreasfunction. 4 representative graphs out of 37 readouts measured showingthe white blood cell count (Unpaired t-test, p=0.8020), Albuminconcentration (Unpaired t-test, p>0.9999), Creatinine concentration(Unpaired t-test, p=0.5490) and Calcium concentration (Unpaired t-test,p=0.811).

Example 15 Systemic Administration of IGF2/but Protects AgainstDexamethasone Induced Muscle Atrophy

Dexamethasone (25 mg/kg i.p.) was administered to 12 weeks old mice for14 days simultaneously with a subcutaneous injection of IGF2/NaB (150ug/kg; 1.2 g/kg) or vehicle (PBS). Muscle function was assessed at day13-14. Grip strength force assessed at day 13, graphs showing bothlimbforce and specific bothlimb force measured on Day 13. Specific bothlimbforce calculated as the ratio of bothlimb force in mN over the weight ing, *** p=0.0003, ***p=0.0004 (Unpaired t-test). Grip strength forceassessed at day 13, graphs showing forelimb force and forelimb specificforce measured on Day 13. Specific forelimb force calculated as theratio of forelimb force in mN over the weight in g, ** p=0.0012,***p=0.0005 (Unpaired t-test). At day 15, mice were euthanized and TAswere collected for histological analysis, graphs showing muscle fibersize distribution assessed using SMASH software. **p=0.054, *p=0.037,and **** p<0.0001 (2-way ANOVA, multiple comparisons).

Example 16 BMP7 Induces Myoblast Proliferation

FIG. 5A) Bar graph quantitation of % EdU+ mouse myoblast in response toBMP7. Myoblast were cultured 48h in the presence of BMP7 at indicateddose. Fresh media and BMP7 was added every 24h, followed by 2 hour EdUpulse and fixation. Mean∓S.D. Significant p-values (Vehicle˜BMP7 0.025ug/mL: 1.21E-07, Vehicle˜BMP7 0.075 ug/mL: 8.05E-07, Vehicle˜BMP7 0.2255ug/mL: 3.37E-03, Vehicle˜BMP7 0.9 ug/mL: 4.99E-02). FIG. 5B) Bar graphquantitation of % EdU+ human myoblast (68 year old caucasian male) inresponse to BMP7. Myoblast were cultured 72h in the presence of BMP7 atindicated dose. Cells were pulsed with EdU for 4 hours before fixation.Mean∓S.D. Significant p-values (Vehicle˜BMP7 1.56 ng/mL: 0.04). (*p<0.05by Welch's One-Tailed T-test, n=2)

Table of data for FIG. 5A - BMP7 Mouse Myoblast Proliferation BMP7 % EdUp-value Vehicle 10.56 —  0.025 ug/mL 18.08 1.21E−07  0.075 ug/mL 17.538.05E−07 0.2255 ug/mL 14.93 3.37E−03  0.45 ug/mL 12.93 n.s.   0.9 ug/mL13.91 4.99E−02   1.8 ug/mL 10.37 n.s.

Table of data for FIG. 5B - BMP7 Human Myoblast Proliferation HumanMyoblast Nuclei Counts p-value Vehicle 2083.5 — 0.78 ng/mL 2144.5 n.s.1.56 ng/mL 2552.5 0.04 3.12 ng/mL 2408 n.s. 6.25 ng/mL 2422.5 n.s. 12.5ng/mL 2706 n.s.   25 ng/mL 2509 n.s.

Example 17 Leucine Enhances BMP7 Mitogenic Activity

FIG. 6A) Bar graph of % EdU+ mouse myoblast cells compared to vehicle.Mouse myoblast were cultured 48h in the presence of indicated factors,followed by a 2 hour EdU pulse prior to fixation. Mean∓S.D. Significantp-values (FM˜BMP7: 0.008 ug/mL: 3.17E-02, FM˜Leucine: 300 uM BMP7: 0.008ug/mL: 2.77E-03, FM˜Leucine: 100 uM BMP7: 0.008 ug/mL: 3.72E-05,FM˜Leucine: 900 uM BMP7: 0.008 ug/mL: 2.52E-03). (*p<0.05 by One-WayANOVA Tukey Honest Significant Difference, n=2-6)

Example 18 Hyaluronic Acid (HA) Enhances BMP7 Mitogenic Activity

FIG. 7A: Bar graph of % EdU+ mouse myoblast cells compared to vehicle.Mouse myoblast were cultured 48h in the presence of indicated factors.EdU pulse and fixation. Mean∓S.D. (*p<0.05 by Student's One-TailedT-Test of increased activity, n=2-6)

Example 19 BMP7 Receptors are Expressed in Human Myoblast

FIG. 8A: Bar graph of BMP7 receptor RNASeq expression in young and agedhuman myoblast (68-69 year old caucasian males) cell lines. Myoblastwere cultured 96h in fusion media. Fresh media was added every 24h,followed by RNA extraction and sequencing. Mean∓SEM. n=3. Expression areexpressed as FPKM.

Example 20 Treatment for Chondrocyte Proliferation in Cartilage Injuryand Osteoarthritis

FIG. 9A: Bar graph showing BMP7 receptors are expressed oncartilage-associated cells. Data derived from Ramilowsky et al., Nature,2015.

Example 21—FGF17-hFcm Promotes Proliferation of Mouse Myoblasts

FIG. 10A) The suspension CHO cells were transiently transfected witheither the empty control plasmid or the FGF17-hFcm encoding plasmid.After four days, the culture supernatants were collected and added intothe culture of mouse myoblast cells for 48 hours, followed by a 2 hourEdU pulse prior to fixation. The percentage of EdU+ mouse myoblaststreated with the culture supernatant of CHO cells expressing FGF17-hFcmis significantly higher than the percentage of EdU+ mouse myoblaststreated with either vehicle control or the culture supernatant of CHOcells expressing the empty control vector (One-Way ANOVA Tukey HonestSignificant Difference, n=2-6). FIG. 10B) The suspension CHO cells weretransiently transfected with the FGF17-hFcm encoding plasmid. After fourdays, the culture supernatants were collected and FGF17-hFcm wasaffinity-purified by Protein A membrane column. The purified FGF17-hFcmwas added into the culture of mouse myoblast cells for 48 hours,followed by a 2 hour EdU pulse and fixation. The percentage of EdU+mouse myoblasts treated with the purified FGF17-hFcm is significantlyhigher than the percentage of EdU+ mouse myoblasts treated with theculture supernatant of CHO cells expressing the empty control vector(One-Way ANOVA Tukey Honest Significant Difference, n=2-6).

Table of data for FIG. 10A Sample % EdU SD p_value Vector controlculture supernatant 7.069 0.354 FGF17-hFcm culture supernatant 30.2852.650 8.88E−6

Table data for FIG. 10B Sample % EdU SD p_value Vehicle control 4.8841.080 Purified FGF17-hFcm 22.761 1.880 2.04E−5

Example 22: FGF17 Mutants Improved Protein Expression Levels in CHOCells

FIG. 11A: SDS-PAGE of culture supernatants from CHO cells transientlytransfected with FGF17-hFcm or its mutants encoding plasmids(FGF17d181-216-hFcm, FGF17d204-216-hFcm, FGF17R204QK207Q-hFcm). Theexpressed FGF17-hFcm proteins and the mutant proteins were indicated bythe white arrowheads. FIG. 11B) The culture supernatants from CHO cellstransiently transfected with different FGF17 encoding plasmids wereadded into the culture of mouse myoblast cells for 48 hours followed by2 hour EdU pulse and fixation. The percentage of EdU+ mouse myoblaststreated with the culture supernatants of CHO cells expressing wild typeFGF17-hFcm or mutants FGF17-hFcm (AA204-216 deletion mutant andR204QK207Q point mutation mutant) is significantly higher than thepercentage of EdU+ mouse myoblasts treated with the culture supernatantof CHO cells expressing the empty control vector (One-Way ANOVA TukeyHonest Significant Difference, n=2-6).

FIG. 11B Sample % EdU SD p_value Vector control 7.069 0.354 FGF17-hFcm30.285 2.650 1.08E−4 FGF17d181-216-hFcm 6.000 0.533 0.99FGF17d204-216-hFcm 39.298 2.002 1.22E−5 FGF17R204QK207Q-hFcm 38.8314.343 1.35E−5

Example 23 FGF17 Mutants Improved Protein Expression Levels in CHO Cells

FIG. 12A) SDS-PAGE of culture supernatants from CHO cells transientlytransfected with FGF17-hFcm or its mutants encoding plasmids(FGF17d197-216-hFcm, FGF17K191AK193AS200A-hFcm). The expressedFGF17-hFcm proteins and the mutant proteins were indicated by the whitearrowheads. FIG. 12B) The culture supernatants from CHO cellstransiently transfected with different FGF17 encoding plasmids wereadded into the culture of mouse myoblast cells for 48 hours, followed by2 hour EdU pulse and fixation. The percentage of EdU+ mouse myoblaststreated with the culture supernatants of CHO cells expressing wild typeFGF17-hFcm or mutants FGF17-hFcm (AA197-216 deletion mutant andK191AK193AS200A point mutation mutant) is significantly higher thanmouse myoblasts treated with the culture supernatant of CHO cellsexpressing the empty control vector (One-Way ANOVA Tukey HonestSignificant Difference, n=2-6).

FIG. 12B Sample % EdU SD p_value Vector control 5.288 0.883 FGF17-hFcm33.806 1.351 5.46E−10 FGF17d197-216-hFcm 28.699 1.926 5.30E−9FGF17K191AK193AS200A-hFcm 33.828 1.434 5.42E−10

Example 24 Human Serum Albumin (HSA) Fusion FGF17 is More Stable inCulture Medium than FGF17 without HSA Fusion Tag

HSA-FGF17 and FGF17 were incubated in culture medium at 37 deg CO2incubator. At different time point (day 0, 1, 3, 5, and 7), an aliquotwas taken and stored at −80 deg. The activity of each sample wasevaluated by mouse myoblast in vitro proliferation assay. The nucleicount at each time point from proliferation assay was normalized to day0 nuclei count.

Example 25 Differential Induction of Myogenic Gene Expression by FGF17in Mouse Myoblasts

Myogenic gene RNA expression (fold change to FM) in response to vehicle(FM) in mouse myoblast cell lines monitored by real-time qPCR. Myoblastwere cultured 48h in fusion media. Mean∓SD. n=3. (Y,Z) Differentialinduction of myogenic gene expression by FGF17 in human myoblastmyoblasts. Quantitation table of myogenic gene RNA expression (foldchange to FM-fusion media (DMEM+2% horse serum)) in response to FM orrh-FGF17 in aged human myoblast cell lines by real-time qPCR. Myoblastwere cultured (B) 48 hours or (C) 72 hours in FM. Mean∓SD. n=3.

Condition Mean SD p-value Gene: Pax7 FM 1.011 0.185 — FGF17 6.118 0.9207.05E−04 Gene: Myf5 FM 1.002 0.071 — FGF17 1.376 0.201 1.69E−02 Gene:Myod1 FM 1.002 0.086 — FGF17 1.230 0.158 n.s. 48 hr MYF5 Condition MeanSD p-value FM 1.04 0.354 FGF17 1.922 0.234 6.06E−03 48 hr MYOD1 FM 1.0010.059 FGF17 0.604 0.095 2.76E−03 48 hr MYOG FM 1.013 0.193 FGF17 2.3530.016 5.77E−03 72 hr MYF5 FM 1.023 0.273 FGF17 0.499 0.234 1.84E−03 72hr MYOD1 FM 1.055 0.423 FGF17 1.627 0.094 n.s. 72 hr MYOG FM 1.092 0.542FGF17 13.124 0.015 6.38E−04

Example 26 FGF17 Receptor is Expressed in Human Myoblasts

FIG. 13A: Bar graph of FGF17 receptor RNASeq expression in young andaged human myoblast cell lines. Myoblast were cultured 96h in fusionmedia with fresh media added every 24h, followed by RNA extraction andsequencing. Mean∓SEM. n=3. Expression values are reported as FPKM.

GeneName Young (n = 6) Old (n = 6) Young_SEM Old_SEM FGFR1 18.069 23.1220.843 1.836

Example 27 Heparin Enhances FGF17 Mitogenic Activity

FIG. 14A: Bar graph of % EdU+ mouse myoblast cells compared to vehicle.Myoblast were cultured 48h in the presence of indicated factors. Freshmedia and factors were added every 24h, followed by 2 hour ECdU pulseand fixation. Mean∓S.D. Table quantitation of % EdU and p-values(*p<0.05 by One-Way ANOVA Tukey Honest Significant Difference, n=2-6)

Condition % EdU SD p-value Vehicle 3.14 0.76 — FGF17: 0.0062 ug/mL 4.810.79 n.s. FGF17: 0.0125 ug/mL 3.02 1.46 n.s. FGF17: 0.025 ug/mL 3.850.59 n.s. Heparin: 0.5 ug/mL 1.77 1.06 n.s. Heparin: 1 ug/mL 3.01 1.27n.s. Heparin: 2 ug/mL 2.85 0.02 n.s. Heparin: 0.5 ug/mL FGF17: 0.0062ug/mL 10.42 3.36 9.19E−04 Heparin: 0.5 ug/mL FGF17: 0.0125 ug/mL 13.011.24 1.09E−06 Heparin: 0.5 ug/mL FGF17: 0.025 ug/mL 28.73 0.29 p <0.4E−22 Heparin: 1 ug/mL FGF17: 0.0062 ug/mL 10.58 1.86 6.07E−04Heparin: 1 ug/mL FGF17: 0.0125 ug/mL 12.88 2.50 1.54E−06 Heparin: 1ug/mL FGF17: 0.025 ug/mL 25.75 1.45 p < 0.4E−22 Heparin: 2 ug/mL FGF17:0.0062 ug/mL 9.74 1.13 4.73E−03 Heparin: 2 ug/mL FGF17: 0.0125 ug/mL14.16 0.96 5.38E−08 Heparin: 2 ug/mL FGF17: 0.025 ug/mL 25.44 0.34 p <0.4E−22

Example 28 Hyaluronic Acid (HA) Enhances FGF17 Mitogenic Activity

FIG. 15A: Bar graph of % EdU+ mouse myoblast cells compared to vehicle.Myoblast were cultured 48h in the presence of indicated factors. Freshmedia and factors were added every 24h, followed by 2 hour EdU pulse andfixation. Mean∓S.D. Table quantitation of % EdU and p-values shown.(*p<0.05 by Student's One-Tailed T-Test of increased activity, n=2-6)

Condition N % Edu SD p-value FM 6 11.162 1.386 — FGF17: 0.025 ug/mL 217.870 2.827 n.s. HA-200K: 0.25% 2 6.672 0.773 n.s. HA-200K: 0.5% 25.561 0.116 n.s. HA-200K: 1% 2 5.276 0.366 n.s. HA-200K: 0.25% FGF17:0.025 ug/mL 2 26.970 9.174 2.10E−02 HA-200K: 0.5% FGF17: 0.025 ug/mL 227.446 6.768 7.53E−03 HA-200K: 1% FGF17: 0.025 ug/mL 2 28.252 2.5112.49E−04

Example 29—Dextran Sulfate (DS) Enhances FGF17 Mitogenic Activity

Bar graph of total EdU+ mouse myoblast cells compared per condition.Myoblast were cultured 48h in the presence of indicated factors. Freshmedia and factors were added every 24h, followed by 2 hour EdU pulse andfixation. Mean∓Standard Deviation (SD) Table quantitation of EdU countsper field and p-values for mouse assay (*p<0.05 by Student's One-TailedT-Test of increased activity, n=3-6, Synergy values <1 are evidence ofeffect). Bar graph of total EdU+ human myoblast cells per condition.Myoblast were cultured 72h in the presence of indicated factors,followed by 4 hour EdU pulse and fixation. Fresh media and factors wereadded every 24h. Mean∓Standard Deviation (SD). Table quantitation of+EdU counts and p-values for human assay. (*p<0.05 by Student'sOne-Tailed T-Test of increased activity, n=3-6, Synergy values <1 areevidence of effect)

TABLE of data Ave. + Synergy, Synergy, EdU Highest Response Condition-mouse N Count SD p-value Single Agent additivity FM 6 165 22 — — —DS10-D3 3 204 23 — — — FGF17 3 476 170 — — — 0.01 ug/mL FGF17 0.1 ug/mL3 1618 508 — — — DS10-D3 + 3 3197 468 3.9E−3 0.145  0.213 0.01 ug/mLFGF17 DS10-D3 + 3 6558 307 3.8E4 0.2247 0.278 0.1 ug/mL FGF17

TABLE of data Synergy, Ave. + Highest Synergy, Condition- EdU SingleResponse human N Counts SD p-value Agent additivity FM 3 205 28 — — —DS10-D2 3 197 18 — — — FGF17 0.1 ug/mL 3 349 62 — — — FGF17 0.1 ug/mL +3 782 85 1.9E−3 0.45 0.70 DS10-D2

Example 30—Intramuscular Administration of FGF17 Promoted theRegeneration of Muscle in BaCl2 Injured Old Mice Model

FIG. 16A) Experiment overview. Intramuscular injection of 1.2% of BaCl2(7 ul/TA) was used to generate chemical injury in the TAs of 78 weeksold mice. FGF17 (500 ng/mL) was administered via intramuscular injectionafter 2h and 48h of muscle injury. FIG. 16B) Quantification of theregenerative index calculated as the number of newly regenerated fibersper mm{circumflex over ( )}2 of injury area. Regenerated fibers wereidentified as fibers with central nuclei,****p<0.0001 (unpaired t-test).FIG. 16C) Histogram showing the fibrotic index calculated as thepercentage of the fibrotic area. *p=0.0186 (unpaired t-test).

Example 31—Systemic Administration of FGF17 Protects AgainstDexamethasone Induced Muscle Atrophy

FIG. 17A) Experiment overview and groups. Dexamethasone (25 mg/kg i.p.)was administered to 12 weeks old mice for 20 days simultaneously with asubcutaneous injection of FGF17 (0.5 mg/kg). Muscle weight was assessedon Day 21. Forelimb grip strength and both limb grip strength weremeasured on Day 7, 13 and 21 FIG. 17B) TAs muscle weight over initialbody weight shown as the percentage change from vehicle. *** p=0.0005,*p=0.0499. Forelimb force measured on Day 21, histogram shows thespecific forelimb force calculated as the ratio of forelimb force in Nover the weight in g, * p=0.0458, **p=0.0014 FIG. 26D17C) Both limbforce measured on Day 21 calculated as the ratio of both limb force in Nover the weight in g ***p=0.001 *p=0.0102. One way Anova corrected formultiple comparisons using Tukey method was used to compare data.

Example 32—Treatment for Chondrocyte Proliferation in Cartilage Injuryand Osteoarthritis and FGF17 Induction of Chondrocyte Proliferation

RNA expression shows FGF17 receptor was expressed oncartilage-associated cells.) Bar graph quantitation of % EdU+ humanchondrocyte in response to FGF17. Chondrocytes were cultured for 48h inthe presence of FGF17 at indicated dose. FGF18 was added at 0.1 ug/mL asa positive control. Fresh media and FGF17 was added every 24h. Table of% EdU+ chondrocyte and p-values depicted. Mean∓S.D. (*p<0.05 by TukeyHonest Significant Difference T-test, n=2-3)

Table FGFR1 RNA Expression (TPM) Cell Type FGFR1 Preadipocyte(Subcutaneous) 54.92 Chondrocyte 152.59 Osteocyte 202.57 Tenocyte 167.01

Table of data Condition n % EdU sd adj-p-val Vehicle 3 0.744 0.247FGF18: 0.1 ug/mL 3 6.039 1.637 1.29E−03 FGF17: 0.1 ug/mL 2 4.675 0.1981.92E−04 FGF17: 0.20 ug/mL 2 9.085 0.094 7.85E−06 FGF17: 0.40 ug/mL 216.773 0.621 5.42E−07

Example 33—Myogenic Activity Measurement Assay In Vitro Mouse MyoblastProliferation Assay

Reduced regeneration from an individual's tissue progenitor cells is ahallmark of age or disease related dysfunction, therefore assays thatmeasure mitogenic capacity in tissue progenitor cells serve as aread-out for potential success of a treatment. Measuring the increasedproliferation rate, degree of differentiation, and cellular survival oftreated mouse or human muscle progenitor cells will provide good basisfor potentially therapeutic regenerative factors for treatingindividuals who have suffered illness, injury, or who possess genetic ordevelopmental defects leading to premature tissue loss, wasting, orweakening.

Mouse muscle progenitor cells (early passage myoblasts) were culturedand expanded in mouse growth medium: Ham's F-10 (Gibco), 20% BovineGrowth Serum (Hyclone), 5 ng/mL FGF2 and 1% penicillin-streptomycin onMatrigel coated plates (1:300 matrigel: PBS), at 37° C. and 5% CO2. Forexperimental conditions, cells were plated at 40,000 cells/well onMatrigel coated 8-well chamber slides in 250-500 μL medium per well(1:100 matrigel: PBS) in mouse fusion medium: DMEM (Gibco)+2% horseserum (Hyclone). One hour after plating, mouse myoblasts were treatedwith 50% respective medias: Mouse myoblasts were cultured for 24 hoursin the above conditions, at 37° C. in 10% CO2 incubator. BrdU (300 μM)in DMSO was added for 2 hours prior to fixation with cold 70% ethanoland stored at 4° C. until staining.

Quantifying Regenerative Index

Following permeabilization in PBS+0.25% Triton X-100, antigen retrievalwas performed. Primary staining was performed with primary antibodiesincluding: a species-specific monoclonal antibody for mouseanti-embryonic Myosin Heavy Chain (eMyHC, hybridoma clone 1.652,Developmental Studies Hybridoma Bank) and Rat-anti-BrdU (Abcam Inc.ab6326). Secondary staining with fluorophore-conjugated,species-specific antibodies (Donkey anti-Rat-488, #712-485-150; Donkeyanti-Mouse-488, #715-485-150. Nuclei are visualized by Hoechst staining.Using the Hoechst stain to tally cell numbers, the percent of cellspositive for BrdU and eMyHC were tabulated and reported.

FGF17-hFcm Promotes Proliferation of Mouse Myoblasts.

Suspension CHO cells were transiently transfected with either the emptycontrol plasmid or the FGF17-hFcm encoding plasmid. After four days, theculture supernatants were collected and added into the culture of mousemyoblast cells for 48 hours, followed by a 2 hour EdU pulse prior tofixation. The percentage of EdU+ mouse myoblasts treated with theculture supernatant of CHO cells expressing FGF17-hFcm is significantlyhigher than the percentage of EdU+ mouse myoblasts treated with eithervehicle control or the culture supernatant of CHO cells expressing theempty control vector (One-Way ANOVA Tukey Honest Significant Difference,n=2-6).

Suspension CHO cells were transiently transfected with the FGF17-hFcmencoding plasmid. After four days, the culture supernatants werecollected and FGF17-hFcm was affinity-purified by Protein A membranecolumn. The purified FGF17-hFcm was added into the culture of mousemyoblast cells for 48 hours, followed by a 2 hour EdU pulse andfixation. The percentage of EdU+ mouse myoblasts treated with thepurified FGF17-hFcm is significantly higher than the percentage of EdU+mouse myoblasts treated with the culture supernatant of CHO cellsexpressing the empty control vector (One-Way ANOVA Tukey HonestSignificant Difference, n=2-6).

Example 34—Myogenic Gene Profiling for Pro-Regenerative Factors

Expression of myogenic factors Pax7, Myf5, Myod1, and Myog are keyindicators of the functional status of muscle progenitor cells. Factorsupregulating of Pax7 and Myf5 indicate rejuvenation of proliferativeprogenitor cells whereas upregulation of Myod1 and Myog are indicativeof muscle myofiber regeneration. A read-out of these gene expressionswill provide potential success for any given polypeptide comprising anFGF17, IGF2, or BMP7 amino acid sequence and an amino acid sequence froma heterologous polypeptide or combinations of an Fibroblast GrowthFactor Receptor agonist and a glycosaminoglycan, an Insulin-like GrowthFactor 1 Receptor (IGF1R) agonist and a short chain fatty acid, and BMPreceptor agonists and mTOR activators and/or glycosaminoglycansdescribed herein. Measuring myogenic genes in mouse or human muscleprogenitor cells treated with factors will provide a goodcharacterization of the therapeutic effect for treating individuals whohave suffered injury, or who possess genetic or developmental defectsleading to premature tissue loss, wasting, or weakening. As a control,the assay will also be performed on proteins purified fromdifferentiated cells, which result in no in myoblast proliferation,cultured in medium conditioned by differentiated cells, or purifiedheparin-associated fractions.

RNA was isolated from each well (RNeasy Mini Kit, Qiagen) and cDNA wasobtained by reverse-transcription (High Capacity Reverse TranscriptionKit, Thermo Fisher Scientific). Real-time quantitative PCR was performedusing QuantStudio3 (Thermo Fisher).

Aged human myoblasts were cultured in well plates. Culturing the cellswith the different medias resulted in differential induction of myogenicgene expression. All factors resulted in changes in at least onemyogenic receptor gene at 48 hours and 72 hours when compared to cellscultured in fusion media, as depicted in Table 4. Cells that had beencultured with IGF2 had increases in levels of MYOG at 48 hours andlevels of MYOD at 72 hours. Table 4: Myogenic transcription factor foldchange increase in myoblasts cultured with IGF2

Con- MYF5 - MYOD1 - MYOG - MYF5 - MYOD1 - MYOG - dition 48 h 48 h 48 h72 h 72 h 72 h FM 1.04 1.001 1.013 1.023 1.055 1.092 IGF2 0.409 0.5195.756 0.708 5.723 0.018

Myogenic Gene Profiling in Human or Mouse Progenitor Cells

Human or mouse muscle progenitor cells will be plated and cultured asdescribed above for myogenic activity testing. One hour after plating,myoblasts will be treated with respective factors. Myoblasts areanalyzed for expression of Pax7, Myf5, Myod1, and Myog to characterizethe regenerative effect of treatment with polypeptides comprising anFGF17, IGF2, or BMP7 amino acid sequence and will be tested tocharacterize the effects an amino acid sequence from a heterologouspolypeptide or combinations of an Fibroblast Growth Factor Receptoragonist and a glycosaminoglycan, an Insulin-like Growth Factor 1Receptor (IGF1R) agonist and a short chain fatty acid, and BMP receptoragonists and mTOR activators and/or glycosaminoglycans.

Example 35—In Vivo Testing of Stem Cell Secreted Factors

Multiple in vivo models of muscle degeneration will be tested. Giventhat polypeptides comprising an FGF17, IGF2, or BMP7 amino acid sequenceand an amino acid sequence from a heterologous polypeptide orcombinations of an Fibroblast Growth Factor Receptor agonist and aglycosaminoglycan, an Insulin-like Growth Factor 1 Receptor (IGF1R)agonist and a short chain fatty acid, and BMP receptor agonists and mTORactivators and/or glycosaminoglycans described herein have regenerativeproperties in in vitro models, these in vivo models will show thatsimilar regenerative and proliferative effects in the context of intactorgan systems.

Acute Injury Model

The experimental groups will be: C57BL/6J male mice, N=18; Young: 12-13week old (3-month-old) mice, n=6; Aged: 77-78 week old (18-month-old)mice, n=12. This design will be used to test any single factoridentified and validated in in vitro assays or polypeptides comprisingan FGF17, IGF2, or BMP7 amino acid sequence and an amino acid sequencefrom a heterologous polypeptide or combinations of an Fibroblast GrowthFactor Receptor agonist and a glycosaminoglycan, an Insulin-like GrowthFactor 1 Receptor (IGF1R) agonist and a short chain fatty acid, and BMPreceptor agonists and mTOR activators and/or glycosaminoglycans.

On Day 0, mice will be weighed and undergo muscle injury with focalinjection of barium chloride (BaCl2, 10 μL, 1.2% w/v in saline,Sigma-Aldrich) in the Tibialis anterior (TA; Day 0) of both the rightand left hindlegs. Injections of vehicle or factor A (0.1 mg/kg) will beco-administered intramuscularly (i.m) following the BaCl2 into the TAinjured hindleg sites, and again 48 hours later on day 2 (i.m.) into theTA injured hindleg sites. Also on day 2, BaCl2 (Ctx; 10 μL, 1.2% w/v insaline, Sigma-Aldrich) was injected into the Gastrocnemius (GA, Day 2,i.m.) muscles of both right and left hind legs. Injections of vehicle ora factor will be sequentially administered (i.m.) following the BaCl2into the TA hindleg sites post-injury, and again 48 hours later on day 4(i.m.) into the GA injured hind leg sites. Bromodeoxyuridine (BrdU) wasbe administered (100 mg/kg, i.p.) once daily for 3 days, day 2-4, beforesacrifice to label proliferating cells.

On day 5, animals will be sacrificed, and animal weight recordedfollowed by collecting 0.5 ml of terminal blood via cardiac puncturewhich was processed to plasma and stored at 80° C. We then perfuse theanimal with 1×PBS, carefully dissect the skin from the GA/TA muscles ofeach hind leg and took photos (prior to excision). After excision ofexclusively the GA or TA muscle, excised tissue is photographed,weighed, then placed into 25% sucrose in PBS at 4° C. for 4 hr rinsed in1×PBS, immersed in Tissue-TEK OCT and rapidly frozen before storing themuscles tissues frozen at 80° C. Cryosectioning and H&E will beperformed to ensure muscle injury site was appropriately visualized.Muscle tissue composition from new skeletal muscle fibers, fibrotictissue, and adipose (fat), will be measured. Muscle regeneration, asdefined as the number of number of new myofibers with centrally locatednuclei per millimeter, fibrosis as defined as the area of fibroticscarring, size of the fibers, as defined as the width and area, adiposetissue, as defined by the amount of fat surrounding the muscle, will bemeasured to assess level of regeneration.

Sarcopenia/Chronic Administration Model

The experimental design is C57BL/6J male mice, N=18; Young: 12-13 weekold (3-month-old) mice, n=6; Aged: 77-78 week old (18-month-old) mice,n=12, as depicted in Table 4. This design can be used to test any singlefactor identified and validated in in vitro assays or complex mixturesof 2 or more factors or synergistic small molecules.

On Day 0, mice will have the following in vivo healthspan measurementswill be performed over 1 day as a baseline for age-based parameters:Weight, running wheel performance, grip strength, and horizontal bar.Each assay should be run for 4 trials per assay per animal. Thesehealthspan assays will be repeated on day −1. After one day of rest onday −9, mice will begin 1×daily injections (0.1 mg/kg) of vehicle orfactor A for the remainder of the experiment until sacrifice (days −8 to+5, 13 days of dosing). On day −4, 6 days after dosing begins, mice willundergo a repeat of the healthspan assays. On day 0, 5 days prior tosacrifice, mice will undergo muscle injury with focal injection ofcardiotoxin (Ctx; 10 pg, Sigma-Aldrich) in the Tibialis anterior (TA;day 0) of the right hindleg only. On day 2, the Gastrocnemius (GA; day2) muscle of the right hind leg will then receive cardiotoxin (Ctx; 10pg, Sigma-Aldrich). BrdU will be administered (100 mg/kg, i.p.) oncedaily for 3 days, day 2-4, before sacrifice. On day +5, prior totake-down, the animals will have an in vivo in capacitance assay run. Onday +5, animals will be sacrificed, and animal weight recorded. We willCollect 0.5 ml of blood via cardiac puncture, process to plasma andstore plasma samples at 80° C. The animals will then be perfused with1×PBS. Carefully dissect the skin from the GA/TA muscles of each hindleg and take photos (prior to excision). After excision of exclusivelythe GA or TA muscle, we will weigh the muscles, then place muscles into25% sucrose in PBS at 4° C. for 4 hours, then rinse the muscles in1×PBS, adding Tissue-TEK OCT and storing the muscles tissues frozen at80° C. Perform cryosectioning and H&E, ensuring muscle injury site isappropriately visualized. Carefully excising the inguinal white adiposetissue (WAT) will be weighed.

Muscle tissue composition, from new skeletal muscle fibers, fibrotictissue, and adipose (fat), will be measured. Muscle regeneration, asdefined as the number of number of new myofibers with centrally locatednuclei per millimeter, fibrosis, as defined as the area of fibroticscarring, size of the fibers, as defined as the width and area, adiposetissue, as defined by the amount of fat surrounding the muscle, will bemeasured to assess level of regeneration. Weights of the animals duringthe duration of treatment, as well as healthspan assays includingperformance on a running wheel (speed, distance, duration), gripstrength, and performance on a horizontal bar will take into account thephenotypic outcomes of treatment of the aged animals systemically withthe polypeptides comprising an FGF17, IGF2, or BMP7 amino acid sequenceand an amino acid sequence from a heterologous polypeptide orcombinations of an Fibroblast Growth Factor Receptor agonist and aglycosaminoglycan, an Insulin-like Growth Factor 1 Receptor (IGF1R)agonist and a short chain fatty acid, and BMP receptor agonists and mTORactivators and/or glycosaminoglycans.

The horizontal bar test will be performed as described previously(Malinowska et al. 2010) at 8 months (n=6 WT, n=7 MPS IIIB) and 10months (n=3 WT, n=4 MPS IIIB) of age. In brief, a 300-mm metal wire, 2mm in diameter, was secured between two posts 320 mm above a paddedsurface. The mouse will be allowed to grip the center of the wire andthe time to fall or reach the side was recorded, and after 2 minutes thetest was stopped. Crossing the bar in x seconds will be scored as 240-x,remaining on the bar will be scored as 120, and falling off the barafter y seconds will be recorded as the value of y. The test will berepeated three times as a practice run followed by a 10-min rest priorto three tests where the score was recorded.

Animals will also have better healthspan outcomes: reduced weight, fatcomposition, scar tissue around muscles, increased running speed,duration, and distance, increased grip strength, and enhancedperformance on the horizontal bar test.

Genetically Obese Muscle Dystrophy Model

Genetically obese (ob/ob) mice will be injected with BaCl2 on day 0 inthe TA muscle. 3 mice will be treated with vehicle only, 3 mice will beinjected with the hPSC factors and 3 mice will be treated with FGF19(positive control) on day 0 and day 2. On day 5, the mice will beeuthanized, the TA muscles perfused with PBS, and dissected. Muscleswill be then analyzed for regenerative index and fibrotic index.

Atrophy Model

The experimental design is C57BL/6J male mice with daily administrationof Vehicle, N=7; dexamethasone (25 mg/kg i.p.), N=6, or dexamethasone+treatment article, N=6, for 20 days. This design can be used to test anysingle factor identified and validated in in vitro assays or complexmixtures of 2 or more factors.

On day 21, animals are sacrificed, and animal weight recorded. 0.5 ml ofblood is collected via cardiac puncture and processed to plasma forstorage of plasma samples at −80° C. The animals are perfused with1×PBS. After carefully dissecting the skin from the GA/TA muscles ofeach hind leg, photos are taken, followed by excision of exclusively theGA or TA muscle, weighing the muscles, then flash freezing in isopentaneat −80 C.

Methods of Testing Muscle Strength. Endurance and Function

Forelimb and Both limb grip strength test: After 30 min acclimation, themice are introduced to the grip strength meter. For forelimb gripstrength, the mice held by the tail are allowed to grasp the grip barwith only its forelimbs. For both limb measurements the mice are placedon the grid and allowed to grasp the grid with both limbs. The forcegenerated by each mouse is calculated as the average of 5-6measurements.

Limb endurance test: Mice are allowed to discover and acclimate therodent treadmill environment through 2 training sessions of 10 minuteseach at 10 m/min on separate days prior to the endurance test. For theendurance test, mice are placed in the individual lanes of the rodenttreadmill. The speed is gradually increased at 2m/min until exhaustionis reached. Exhaustion is defined as a mouse staying on a grillelectrified to deliver a shock of 2 Hz, intensity 5 for 3-5 seconds.

In vivo tetanic force measurement: Mice are under anesthesia usingregulated delivery of isoflurane during the whole process. Followinganesthetization, the animal is placed onto a heated chamber with thefoot secured on the foot pedal of an Aurora force transducer. The 2electrodes are placed specifically to stimulate the sciatic nerve. Theforce generated by the ankle torsion of the animal's hind limb, asopposed to direct force is measured in response to a series ofstimulation that includes 50, 100, 150 and 200 Hz.

In situ tetanic force measurement: This experiment is performed usingAurora force measurement. Mice are under anesthesia during the wholeprocess. A small incision in the skin around the Anterior Tibialisexposes the Achilles tendon which is connected via surgical suture tothe Aurora force transducer through a hook. The force generated by themuscle in response to a series of stimulation that includes 50, 100, 150and 200 Hz by 2 electrodes placed on the anterior tibialis is recorded.

Example 36—Mitogenic Polypeptide Stability In Vivo Assayed byBioavailability and Pharmacokinetics Bioavailability in Tissues

The bioavailability of the therapeutic polypeptides will be assessed inthe target tissues in young mice (10-12 weeks old) and old mice (78weeks old). For this experiment, 1 cohort of young mice (10-12 weeksold; N=24) and 1 cohort of old mice (78 weeks old; N=24) will receive 1subcutaneous (SC) injection of a therapeutic composition. 4 young mice(10-12 weeks old; N=6) and 4 old mice (78 weeks old; N=6) will receive 1SC injection of Vehicle and used as control. 4 mice from each cohortwill be euthanized after 30 minutes, 1 hour, 1.5 hours, 2 hours, or 4hours. At each time point blood will be collected by heart puncturefollowed by harvesting select tissues, such as the tibialis anterior,gastrocnemius, quadriceps, heart and diaphragm. The detection andquantitation of the administered therapeutic polypeptides will bedetected by enzyme-linked immunosorbent assay (ELISA). The level oftherapeutic polypeptides will be compared to the samples collected frommice injected with vehicle to determine tissue level bioavailability.

Pharmacokinetics of Engineered Mitogenic Polypeptides

Murine pharmacokinetics (PK) represents the absorption, distribution,metabolism, and elimination of drugs from the body. The pharmacokineticprofile of the therapeutic polypeptides will be determined in young mice(10-12 weeks old) and old mice (78 weeks old). For this experiment, 2routes of administration will be investigated, including SC andintravenous (IV) injection in both young (10-12 weeks old) and old mice(78 weeks old). Six time points for each group 5, 15, 30, 60, 90 and 120minutes will be assessed using end-point or serial sampling. At least 4animals will be used for each time point/group/route. Engineeredmitogenic polypeptides concentrations in the samples will be measured byLC-MS/MS or ELISA. Various pharmacokinetics will be calculated as wellas the absorption/elimination dynamics following different routes ofadministration.

Example 37—Additional Tests for Regenerative Factors

Mechanistic insight into polypeptides comprising an FGF17, IGF2, or BMP7amino acid sequence and an amino acid sequence from a heterologouspolypeptide or combinations of an Fibroblast Growth Factor Receptoragonist and a glycosaminoglycan, an Insulin-like Growth Factor 1Receptor (IGF1R) agonist and a short chain fatty acid, and BMP receptoragonists and mTOR activators and/or glycosaminoglycans combinationspathway of action will be gained by establishing and screening against apanel of assays for cellular age. Assays include measurements ofreactive oxygen species (ROS) production or tolerance cytoplasmicallyand in the mitochondria, telomerase activity, measurements ofproteostasis capacity via lysosomal, autophagy, and proteasomal routes,epigenetic re-patterning, and cellular energy balance (e.g., ATP/ADP andNAD/NADH ratios). Many of these assays leverage high-throughputautomated microscopy to make these measurements in a variety of celltypes, including fibroblast, endothelial cells, mesenchymal stem cells,and chondrocytes. Collectively these metrics can inform both the pathwayand the mechanisms by which the heparin-associated hPSC secretome or itsindividual components enact their regenerative effects. These deepprofile vectors can be crucial for approaching combinations of factorsrationally, and for machine learning predictions.

To test the cellular effects of secretomes toward reversing thehallmarks of aging, high-throughput automated imaging and quantificationof single cells to achieve deep population level statistical power canbe employed. Cellular component state profiles of Young, Aged, andAged+Treatment in human fibroblasts and epithelial cells, myoblasts,mesenchymal stem cells, chondrocytes, and neural progenitor cells willbe compared. Some examples of tests and methods include:

Epigenetic reprogramming: repressive mark H3K9me3, theheterochromatin-associated protein HP1γ, nuclear lamina support proteinLAP2α.

Nuclear membrane Folding/Blebbing: immunofluorescence of the nuclearmembrane protein Lamin A/C.

Proteolytic Activity: Cleavage of fluorescent-tagged chymotrypsin likesubstrate corresponds to proteasome 20S core particle activity. Wellswill be first stained with PrestoBlue Cell Viability dye (LifeTechnologies) for 10 minutes. Well signals will be read using a TECANfluorescence plate reader as a measure of cell count. Then cells will bewashed with HBSS/Ca/Mg before switching to original media containing thechymotrypsin like fluorogenic substrate LLVY-R110 (Sigma) which iscleaved by the proteasome 20S core particle. Cells will be thenincubated at 37° C. in 5% CO2 for 2 hours before signals will be againread on the TECAN fluorescence plate reader. Readings will be thennormalized by PrestoBlue cell count.

Formation of autophagosomes: Autophagosome number and volume will bemeasured by staining with CellTracker Deep Red (Sigma). The cells willbe then incubated at 37° C. in 5% CO2 for 20 minutes, washed 2 timesusing HBSS/Ca/Mg, and stained for 15 minutes using CellTracker Deep Redcell labeling dye. Cells will be then switched to HBSS/Ca/Mg for singlecell imaging using the Operetta High Content Imaging System (PerkinElmer).

Energy Metabolism: ATP in the cells is measured using colorimetric assayusing an ATP assay kit (ab83355; Abcam, Cambridge, MA) followingmanufacturer's instructions. Cells will be washed in cold phosphatebuffered saline and homogenized and centrifuged to collect thesupernatant. The samples will be loaded with assay buffer in triplicate.ATP reaction mix and background control (50 μL) is added to the wellsand incubated for 30 min in dark. The plate is read at OD 570 nm usingSpectraMax M2e (Molecular Devices, Sunnyvale, CA). The mean opticaldensity is used to estimate of the intracellular ATP concentrationrelative to the standard curve.

Mitochondrial Activity: To measure Mitochondria Membrane Potential,cells will be washed twice with Ham's F10 (no serum or pen/strep).Subsequently, MuSCs will be stained with MitoTracker Green FM(ThermoFisher, M7514) and DAPI for 30 minutes at 37° C., washed threetimes with Ham's F10, and analyzed using a BD FACSAria III flowcytometer.

Mitochondrial ROS Measurement. Cells will be washed with HBSS/Ca/Mg andthen switched to HBSS/Ca/Mg containing MitoSOX (Thermo), a live cellpermeant fluorogenic dye that is selectively targeted to mitochondriaand fluoresces when oxidized by superoxide. Cells will be incubated for10 minutes at 37° C. in 5% CO2. Cells will be then washed twice withHBSS/Ca/Mg, and stained for 15 minutes using CellTracker Deep Red.Finally, cells will be imaged in fresh HBSS/Ca/Mg using the OperettaHigh Content Imaging System (Perkin Elmer).

Deregulated Nutrient Sensing: levels of SIRT1 will be measured.

Senescence: Senescence-associated beta-galactosidase staining ismeasured in cells washed twice with PBS then fixed with 15%Paraformaldehyde in PBS for 6 minutes. Cells will be rinsed 3 times withPBS before staining with X-gal chromogenic substrate, which is cleavedby endogenous Beta galactosidase. Plates will be kept in the stainingsolution, Parafilmed, to prevent from drying out, and incubatedovernight at 37° C. with ambient CO2. The next day, cells will be washedagain with PBS before switching to a 70% glycerol solution for imagingunder a Leica brightfield microscope.

Secretome of the cells: Mass-Spec or O-Link for inflammatory cytokinesprofiles.

Soft Tissue Deposition: Immunofluorescence for SOX9, MMP3, MMP13, andCOL2A1 expression, the decrease of which is characterized by cartilageloss, pain, cleft-lip, and joint destruction.

Example 38—The Purified IGF2-hFcm Promoted Differentiation of MyoblastCells

Suspension CHO cells were transiently transfected with the IGF2-hFcmencoding plasmid. After four days, the culture supernatants werecollected and IGF2-hFcm was affinity-purified by Protein A membranecolumn. The purified IGF2-hFcm was added into the culture of humanmyoblast cells. Myosin heavy chain (MyHC) was immunostained and imagedby a fluorescent microscope. After quantification of the stained MyHC,the percentage area of MyHC was calculated as the percent of pixelswithin the field that are illuminated above background in the stainedchannel. The percentage of EdU of mouse myoblasts treated with thepurified IGF2-hFcm is significantly higher than the percentage of EdU ofmouse myoblasts treated with the culture supernatant of CHO cellsexpressing the empty control vector. Significance was determined by ap-value less than 0.05 by the one-way ANOVA Tukey Honest SignificantDifference test.

TABLE 5 IGF2 promoted differentiation of myoblast cells Condition % MyHCSD p_value Vehicle control 1.787 0.186  33 nM IGF2-hFcm 3.734 0.7900.012  66 nM IGF2-hFcm 5.922 0.795 3.20E−05 133 nM IGF2-hFcm 7.568 0.5381.46E−06

This example found that the IGF2-fusion protein was able to induce cellproliferation. The IGF2-fusion protein shares in vitro properties withthe HAPs, which is suggestive of shared in vivo properties.

Example 39—Modelling Treatment of a Muscular Dystrophy with an IGF2Composition In Vitro

Muscular dystrophies (MD) encompass a variety of muscular degenerationdiseases typically due to genetic mutations in genes encoding proteinsresponsible for forming and stabilizing skeletal muscle. The phenotypicconsequence of these genetic mutations is the progressive loss of musclemass and strength over time, similar to sarcopenia but with differentunderlying causes. As HAPs provided phenotypic improvements onsarcopenic muscle, we tested for similar improvements in a model for MD.

IGF2 was tested individually for its ability to promote proliferationand/or fusion of human muscle progenitor cells from an individual withmyotonic dystrophy type 1 (hMD)—a muscular dystrophy caused by mutationsin the DMPK1 gene. The effect of IGF2 on myogenic activity was assayedin biological triplicate across a range of concentrations centeredaround expected physiological levels by adding each factor to hMDmyoblasts for 72 hours with daily media changes (DMEM+2% horse serum)and a second pulse of factors at the first media change. After 72 or 96hours, cells were pulsed for 2-5 hours with EdU (30 uM), ethanol fixed,stained with Hoescht 3342, immunostained for proliferation—as measuredby the percent of cells staining positive for EdU (% EdU)—, andimmunostained for differentiation—as measured by the increase incellular area staining positive for embryonic myosin heavy chain (%eMyHC) relative to the negative controls, which received media andvehicle only. Wells were imaged on a Keyence BZ-100 at 4×, the imagesquantified in Cell Profiler, and the statistics were computed in R.Additionally, RNA was extracted from myoblast and select transcriptabundances quantified by qPCR. depicts IGF2 treatment promotedproliferation and differentiation respectively in DM1 human myoblast (32year old caucasian female) cells. depict IGF2 enhanced MYH3, CKM, andATP1B1 expression in DM1 human myoblast (32 year old caucasian female)cells.

Example 40—Systemic Administration of Therapeutic Polypeptides ReversesSarcopenia and Protects from Muscle Injury

A daily subcutaneous injection of therapeutic polypeptides or vehicleonly is administered to 78 week old mice for 14 days. IGF2 is injectedat a concentration of 100-1000 μg/kg. In some experiments, treatmentgroups receive a single therapeutic factor while in other experiments,treatment groups receive a combination of factors. At 7 days, musclefunction is assessed using forelimb grip strength and both gripstrength. On day 12, 13 and 14, groups 1 and 2 are injected with BrdUintraperitoneally. On days 13-15, all mice are assessed for gripstrength and an endurance test to determine max distance and max speedand tetanic force.

At 15 days, mice in groups 1 and 2 are euthanized and the muscles areanalyzed for markers of proliferation and fibrosis. At 15 days, anintramuscular injection of 1.2% of BaCl2 (7 ul/TA) is used to generatechemical injury in the TAs of group 3 and group 4. Mice from groups 3and 4 continue to receive a therapeutic polypeptide injectedsubcutaneously on days 15-21. They also receive BrdU injectionsintraperitoneally on days 19, 20, and 21. On day 21, the TA muscles aretested for in situ tetanic force. The TA muscles are dissected andassessed for signs of proliferation and fibrosis.

Example 41—Systemic Administration of Fusion Polypeptides ReversedInduced Muscle Atrophy

12-week-old mice are divided into 3 treatment groups: group 1 whichreceives injections only of the vehicle, group 2 which receivesinjections of dexamethasone, and group 3 which receives injections ofdexamethasone and IGF2 fusion polypeptide. Dexamethasone (25 mg/kg i.p.)is administered for 14 days simultaneously with a subcutaneous injectionof IGF2 fusion polypeptide.

At 7 days, mice are assessed for forelimb and both limb grip strength.At days 13-15, mice are assessed for grip strength, in vivo tetanicforce, and undergo a treadmill endurance test to determine max speed andmax distance.

Example 42—Systemic Administration of IGF2 Fusion Polypeptide Predictedto Improve Muscle Atrophy in Genetically Obese Mice

Thirteen-week old genetically obese mice (ob/ob) will be injectedsubcutaneously with an IGF2 fusion polypeptide for 14 days. At day 7,forelimb and both grip strength will be measured. BrdU is injected ondays 12, 13 and 14. On days 13, 14 and 15, forelimb and both limb gripstrength and in vivo tetanic force will be tested, and an endurance testto determine max distance and max speed is performed. At 14 days, themice will be euthanized, and the TA muscles dissected. Muscle weight andproliferation will be analyzed.

Example 43—Systemic Administration of IGF2 Fusion Polypeptide Predictedto Reverse of Slow Down Dystrophic Features in 70 Weeks Old mdx Mice

Another class of human myopathies in need of treatment are the geneticabnormality induced muscular dystrophies, among which Duchenne musculardystrophy is a rare but fatal case. Old genetically dystrophic (mdx)mice (>15 month old) show similar features to the human Duchennemuscular dystrophy (DMD), notably, a decrease in muscle regenerationleading to muscle wasting. Treatment with IGF2 fusion polypeptide canreverse the dystrophic features of old mdx mice. During the acclimationperiod, the weight, Forelimb and both limb grip strength as well as invivo tetanic force will be assessed to determine the baseline strengthof each mouse. 70 week dystrophic mice (mdc) are injected with the IGF2fusion polypeptide subcutaneously for 14 days. At day 7, forelimb andboth grip strength are measured. BrdU is injected on days 12, 13 and 14.On days 13, 14, and 15, forelimb and both limb grip strength and in vivotetanic force are tested, and an endurance test to determine maxdistance and max speed is performed. The right tibialis anterior andgastrocnemius will be collected, immersed in Tissue-TEK OCT and thenflash frozen in chilled isopentane bath precooled in liquid nitrogen andstored at −80° C. Tissue will be sectioned and stained for Laminin todetermine the cross sectional area (CSA) of muscle fibers, for eMyHC tomeasure new fiber formation and for BrdU to assess the proliferationrate. The left anterior tibialis and gastrocnemius will be collected andflash frozen in liquid nitrogen for molecular analysis that include qPCRand western blot.

IGF2 is predicted to be effective at a concentration of 10-200 ug/kg.

Example 44—Systemic Administration of IGF2 Fusion Polypeptide Predictedto Improve the Dystrophic Features in 6 Week Old Mice

Between 3-6 weeks old, the skeletal muscle of mdx mice undergoes severenecrosis followed by an increase in the activation of satellite cells topromote muscle regeneration. Treatment with IGF2 fusion polypeptidedescribed herein can improve the regeneration process and thereforemuscle health. During the acclimation period, the weight, Forelimb andboth limb grip strength as well as in vivo tetanic force will beassessed to determine the baseline strength of each mouse. 6 week olddystrophic mice (mdx) are injected with the IGF2 fusion polypeptidesubcutaneously for 14 days. At day 7, forelimb and both grip strengthare measured. BrdU is injected on days 12, 13 and 14. On days 13, 14 and15, forelimb and both limb grip strength and in vivo tetanic force aretested, and an endurance test to determine max distance and max speed isperformed.

Mice will be euthanized. The right tibialis anterior and gastrocnemiuswill be collected, immersed in Tissue-TEK OCT and then flash frozen inchilled isopentane bath precooled in liquid nitrogen and stored at −80°C. Tissue will be sectioned and stained for Laminin to determine thecross sectional area (CSA) of muscle fibers, for eMyHC to measure newfiber formation and for BrdU to assess the proliferation rate. The leftanterior tibialis and gastrocnemius will be collected and flash frozenin liquid nitrogen for molecular analysis that include qPCR and westernblot.

IGF2 is predicted to be effective at a concentration of 10-200 μg/kg.

Example 45—Treatment for Chondrocyte Proliferation in Cartilage Injuryand Osteoarthritis

Cartilage can become damaged as a result of a sudden injury or due togradual wear and tear or inflammation leading to disease states (e.g.osteoarthritis). Chondrocytes secrete the cartilage matrix andpreadipocytes, osteocytes and tenocytes are all cell types associatedwith cartilage.

Preadipocytes, chondrocytes, osteocytes and tenocytes were cultured inwell plates. RNA was isolated from each well (RNeasy Mini Kit, Qiagen)and cDNA was obtained by reverse-transcription (High Capacity ReverseTranscription Kit, Thermo Fisher Scientific). Real-time quantitative PCRwas performed using QuantStudio3 (Thermo Fisher).

These cartilage-associated cells expressed receptors for polypeptidescomprising an FGF17, IGF2, or BMP7 amino acid sequence and an amino acidsequence from a heterologous polypeptide or combinations of anFibroblast Growth Factor Receptor agonist and a glycosaminoglycan, anInsulin-like Growth Factor 1 Receptor (IGF1R) agonist and a short chainfatty acid, and BMP receptor agonists and mTOR activators and/orglycosaminoglycans. Subcutaneous preadipocytes, chondrocytes, osteocytesand tenocytes all expressed one or more receptors, indicating that thesepolypeptides comprising an FGF17, IGF2, or BMP7 amino acid sequence andan amino acid sequence from a heterologous polypeptide or combinationsof an Fibroblast Growth Factor Receptor agonist and a glycosaminoglycan,an Insulin-like Growth Factor 1 Receptor (IGF1R) agonist and a shortchain fatty acid, and BMP receptor agonists and mTOR activators and/orglycosaminoglycans may be able to affect cartilage loss and theprogression of joint related injury or disease recovery.

Example 46—Clinical Testing of Therapeutic Compositions

The purpose of this study is to determine the safety, tolerability, andpharmacokinetics of repeat dosing with multiple dose levels ofpolypeptides comprising an FGF17, IGF2, or BMP7 amino acid sequence andan amino acid sequence from a heterologous polypeptide or combinationsof an Fibroblast Growth Factor Receptor agonist and a glycosaminoglycan,an Insulin-like Growth Factor 1 Receptor (IGF1R) agonist and a shortchain fatty acid, and BMP receptor agonists and mTOR activators and/orglycosaminoglycans in healthy individuals or individuals diagnosed withsarcopenia, a muscular dystrophy, or recovery from surgery. In certainembodiments, the muscular dystrophy is myotonic dystrophy. In addition,this study will generate data on the physical function, skeletal musclemass and strength resulting from treatment with IGF2 fusion polypeptidesin such individuals. Individuals will be administered placebo or IGF2fusion polypeptide compositions and monitored for 25 weeks of study. Thefollowing primary and secondary outcome measures will be assessed:

Primary Outcome Measures:

Safety and tolerability as assessed by various measures such as percentof adverse events per study arm.

Secondary Outcome Measures:

Plasma Pharmacokinetics (Cmax, Tmax, AUC) [Plasma at 0.5, 1, 1.5, 2, 4,6, 8, 12 and 24 hrs after dosing.]

Short Physical Performance Battery (SPPB). Change from baseline to week25.

10-meter walk test. Change from baseline to week 25.

Change in total lean body mass and appendicular skeletal muscle indexmeasured by Dual-energy X-ray Absorptiometry (DEXA) from baseline toweek 25.

Inclusion Criteria:

Diagnosis of sarcopenia, a muscular dystrophy, or recovery from surgery;Low muscle mass as confirmed by DXA; Low gait speed; SPPB score lessthan or equal to 9; Weigh at least 35 kg; with adequate dietary intakeas determined by patient interview. Independently ambulatory to 10meters.

Protocol

Patients will be i.v.-administered placebo (5% dextrose solution) ortreatment article (in 5% dextrose). Starting on day 1, week 1 andrepeated every week (day one of weeks 1 through 25). At the end of week13 and 25 patients will be assessed by the above methods forimprovement. Doses will be selected from a traditional 3+3 design, andselected as the top two-doses that lack dose-limiting toxicity.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention.

All publications, patent applications, issued patents, and otherdocuments referred to in this specification are herein incorporated byreference as if each individual publication, patent application, issuedpatent, or other document was specifically and individually indicated tobe incorporated by reference in its entirety. Definitions that arecontained in text incorporated by reference are excluded to the extentthat they contradict definitions in this disclosure.

TABLE 1 Secretory Signal Sequences Seq Se- ID quence No Name DescriptionSequence  1 spFGF- Human FGF-17 ATGGGAGCCGCCCGCCTGCTGC 17 secretoryCCAACCTCACTCTGTGCTTACA signal GCTGCTGATTCTCTGCTGTCAA peptide nucleotide sequence  2 spTHBS1 Human THBS1 ATGGGGCTGGCCTGGGGACTAG secretory GCGTCCTGTTCCTGATGCATGT signal  GTGTGGCACC peptide nucleotide  sequence 3 spIGF- Human IGF-2  ATGGGAATCCCAATGGGGAAGT 2 secretoryCGATGCTGGTGCTTCTCACCTT signal  CTTGGCCTTCGCCTCGTGCTGC peptide  ATTGCTnucleotide sequence  4 spBMP- Human BMP-7 ATGCACGTGCGCTCACTGCGAG 7secretory  CTGCGGCGCCGCACAGCTTCGT signal  GGCGCTCTGGGCACCCCTGTTC peptideCTGCTGCGCTCCGCCCTGGCC nucleotide  sequence  5 spALB Human ATGAAGTGGGTAACCTTTATTT Albumin CCCTTCTTTTTCTCTTTAGCTC secretory GGCTTATTCC signal  peptide nucleotide  sequence  6 spAZU1 Human ATGACCCGGCTGACAGTCCTGG Azurocidin CCCTGCTGGCTGGTCTGCTGGC secretory GTCCTCGAGGGCC signal  peptide nucleotide  sequence  7 spBM40 Human ATGAGGGCCTGGATCTTCTTTC osteonectin TCCTTTGCCTGGCCGGGAGGGC secretory TCTGGCAGCA signal  peptide nucleotide  sequence  8 spGAU Gaussia ATGGGAGTCAAAGTTCTGTTTG luciferase CCCTGATCTGCATCGCTGTGGC secretory CGAGGCC signal  peptide nucleotide  sequence  9 spFGF- Human FGF-17MGAARLLPNLTLCLQLLILCCQ 17 secretory  signal  peptide amino acid sequence 10 spTHBS1 Human THBS1 MGLAWGLGVLFLMHVCGT secretory  signal peptide amino acid  sequence 11 spIGF- Human IGF-2 MGIPMGKSMLVLLTFLAFASCC 2 secretory IA signal  peptide  amino acid sequence 12 spBMP- Human BMP-7 MHVRSLRAAAPHSFVALWAPLF 7 secretory LLRSALA signal  peptide amino acid  sequence 13 spALB Human MKWVTFISLLFLFSSAYS Albumin secretory  signal  peptide amino acid sequence 14 spAZU1 Human  MTRLTVLALLAGLLASSRA Azurocidin secretory signal  peptide amino acid  sequence 15 spBM40 Human  MRAWIFFLLCLAGRALAAosteonectin secretory  signal  peptide amino acid  sequence 16 spGAUGaussia  MGVKVLFALICIAVAEA luciferase secretory  signal  peptideamino acid sequence

TABLE 2 Therapeutic Sequences Seq Sequence ID No Name DescriptionSequence 17 FGF-17 Full length of human ACTCAGGGGGAGAATCACCCGTCTCCTAAFGF-17 nucleotide TTTTAACCAGTACGTGAGGGACCAGGGCG sequenceCCATGACCGACCAGCTGAGCAGGCGGCAG ATCCGCGAGTACCAACTCTACAGCAGGACCAGTGGCAAGCACGTGCAGGTCACCGGGC GTCGCATCTCCGCCACCGCCGAGGACGGCAACAAGTTTGCCAAGCTCATAGTGGAGAC GGACACGTTTGGCAGCCGGGTTCGCATCAAAGGGGCTGAGAGTGAGAAGTACATCTGT ATGAACAAGAGGGGCAAGCTCATCGGGAAGCCCAGCGGGAAGAGCAAAGACTGCGTGT TCACGGAGATCGTGCTGGAGAACAACTATACGGCCTTCCAGAACGCCCGGCACGAGGG CTGGTTCATGGCCTTCACGCGGCAGGGGCGGCCCCGCCAGGCTTCCCGCAGCCGCCAG AACCAGCGCGAGGCCCACTTCATCAAGCGCCTCTACCAAGGCCAGCTGCCCTTCCCCAA CCACGCCGAGAAGCAGAAGCAGTTCGAGTTTGTGGGCTCCGCCCCCACCCGCCGGACCA AGCGCACACGGCGGCCCCAGCCCCTCACG 18 FGF-Human FGF-17 AA204- ACTCAGGGGGAGAATCACCCGTCTCCTAA 17d204-216 deletion mutant TTTTAACCAGTACGTGAGGGACCAGGGCG 216nucleotide sequence CCATGACCGACCAGCTGAGCAGGCGGCAGATCCGCGAGTACCAACTCTACAGCAGGAC CAGTGGCAAGCACGTGCAGGTCACCGGGCGTCGCATCTCCGCCACCGCCGAGGACGGC AACAAGTTTGCCAAGCTCATAGTGGAGACGGACACGTTTGGCAGCCGGGTTCGCATCA AAGGGGCTGAGAGTGAGAAGTACATCTGTATGAACAAGAGGGGCAAGCTCATCGGGAA GCCCAGCGGGAAGAGCAAAGACTGCGTGTTCACGGAGATCGTGCTGGAGAACAACTAT ACGGCCTTCCAGAACGCCCGGCACGAGGGCTGGTTCATGGCCTTCACGCGGCAGGGGC GGCCCCGCCAGGCTTCCCGCAGCCGCCAGAACCAGCGCGAGGCCCACTTCATCAAGCG CCTCTACCAAGGCCAGCTGCCCTTCCCCAACCACGCCGAGAAGCAGAAGCAGTTCGAGT TTGTGGGCTCCGCCCCCACC 19 FGF-HumanFGF-17AA181- ACTCAGGGGGAGAATCACCCGTCTCCTAA 17d181-216 deletion mutant TTTTAACCAGTACGTGAGGGACCAGGGCG 216nucleotide sequence CCATGACCGACCAGCTGAGCAGGCGGCAGATCCGCGAGTACCAACTCTACAGCAGGAC CAGTGGCAAGCACGTGCAGGTCACCGGGCGTCGCATCTCCGCCACCGCCGAGGACGGC AACAAGTTTGCCAAGCTCATAGTGGAGACGGACACGTTTGGCAGCCGGGTTCGCATCA AAGGGGCTGAGAGTGAGAAGTACATCTGTATGAACAAGAGGGGCAAGCTCATCGGGAA GCCCAGCGGGAAGAGCAAAGACTGCGTGTTCACGGAGATCGTGCTGGAGAACAACTAT ACGGCCTTCCAGAACGCCCGGCACGAGGGCTGGTTCATGGCCTTCACGCGGCAGGGGC GGCCCCGCCAGGCTTCCCGCAGCCGCCAGAACCAGCGCGAGGCCCACTTCATCAAGCG CCTCTACCAA 20 FGF-17 HumanFGF-17ACTCAGGGGGAGAATCACCCGTCTCCTAA R204Q R204Q K207Q mutantTTTTAACCAGTACGTGAGGGACCAGGGCG K207Q nucleotide sequenceCCATGACCGACCAGCTGAGCAGGCGGCAG ATCCGCGAGTACCAACTCTACAGCAGGACCAGTGGCAAGCACGTGCAGGTCACCGGGC GTCGCATCTCCGCCACCGCCGAGGACGGCAACAAGTTTGCCAAGCTCATAGTGGAGAC GGACACGTTTGGCAGCCGGGTTCGCATCAAAGGGGCTGAGAGTGAGAAGTACATCTGT ATGAACAAGAGGGGCAAGCTCATCGGGAAGCCCAGCGGGAAGAGCAAAGACTGCGTGT TCACGGAGATCGTGCTGGAGAACAACTATACGGCCTTCCAGAACGCCCGGCACGAGGG CTGGTTCATGGCCTTCACGCGGCAGGGGCGGCCCCGCCAGGCTTCCCGCAGCCGCCAG AACCAGCGCGAGGCCCACTTCATCAAGCGCCTCTACCAAGGCCAGCTGCCCTTCCCCAA CCACGCCGAGAAGCAGAAGCAGTTCGAGTTTGTGGGCTCCGCCCCCACCCaGCGGACCc AGCGCACACGGCGGCCCCAGCCCCTCACG 21 FGF-Human FGF17 AA197- ACTCAGGGGGAGAATCACCCGTCTCCTAA 17d197-216 deletion mutant TTTTAACCAGTACGTGAGGGACCAGGGCG 216nucleotide sequence CCATGACCGACCAGCTGAGCAGGCGGCAGATCCGCGAGTACCAACTCTACAGCAGGAC CAGTGGCAAGCACGTGCAGGTCACCGGGCGTCGCATCTCCGCCACCGCCGAGGACGGC AACAAGTTTGCCAAGCTCATAGTGGAGACGGACACGTTTGGCAGCCGGGTTCGCATCA AAGGGGCTGAGAGTGAGAAGTACATCTGTATGAACAAGAGGGGCAAGCTCATCGGGAA GCCCAGCGGGAAGAGCAAAGACTGCGTGTTCACGGAGATCGTGCTGGAGAACAACTAT ACGGCCTTCCAGAACGCCCGGCACGAGGGCTGGTTCATGGCCTTCACGCGGCAGGGGC GGCCCCGCCAGGCTTCCCGCAGCCGCCAGAACCAGCGCGAGGCCCACTTCATCAAGCG CCTCTACCAAGGCCAGCTGCCCTTCCCCAACCACGCCGAGAAGCAGAAGCAGTTCGAG 22 FGF-17 Human FGF-17 K191AACTCAGGGGGAGAATCACCCGTCTCCTAA K191A K193A S200A mutantTTTTAACCAGTACGTGAGGGACCAGGGCG K193A nucleotide sequenceCCATGACCGACCAGCTGAGCAGGCGGCAG S200A ATCCGCGAGTACCAACTCTACAGCAGGACCAGTGGCAAGCACGTGCAGGTCACCGGGC GTCGCATCTCCGCCACCGCCGAGGACGGCAACAAGTTTGCCAAGCTCATAGTGGAGAC GGACACGTTTGGCAGCCGGGTTCGCATCAAAGGGGCTGAGAGTGAGAAGTACATCTGT ATGAACAAGAGGGGCAAGCTCATCGGGAAGCCCAGCGGGAAGAGCAAAGACTGCGTGT TCACGGAGATCGTGCTGGAGAACAACTATACGGCCTTCCAGAACGCCCGGCACGAGGG CTGGTTCATGGCCTTCACGCGGCAGGGGCGGCCCCGCCAGGCTTCCCGCAGCCGCCAG AACCAGCGCGAGGCCCACTTCATCAAGCGCCTCTACCAAGGCCAGCTGCCCTTCCCCAA CCACGCCGAGGctCAGGcaCAGTTCGAGTTTGTGGGCgCtGCCCCCACCCGCCGGACCAAG CGCACACGGCGGCCCCAGCCCCTCACG 23 FGF-17-Full length of human ACTCAGGGGGAGAATCACCCGTCTCCTAA linker1-FGF-17- linker1- hFcm TTTTAACCAGTACGTGAGGGACCAGGGCG hFcmnucleotide sequence CCATGACCGACCAGCTGAGCAGGCGGCAGATCCGCGAGTACCAACTCTACAGCAGGAC CAGTGGCAAGCACGTGCAGGTCACCGGGCGTCGCATCTCCGCCACCGCCGAGGACGGC AACAAGTTTGCCAAGCTCATAGTGGAGACGGACACGTTTGGCAGCCGGGTTCGCATCA AAGGGGCTGAGAGTGAGAAGTACATCTGTATGAACAAGAGGGGCAAGCTCATCGGGAA GCCCAGCGGGAAGAGCAAAGACTGCGTGTTCACGGAGATCGTGCTGGAGAACAACTAT ACGGCCTTCCAGAACGCCCGGCACGAGGGCTGGTTCATGGCCTTCACGCGGCAGGGGC GGCCCCGCCAGGCTTCCCGCAGCCGCCAGAACCAGCGCGAGGCCCACTTCATCAAGCG CCTCTACCAAGGCCAGCTGCCCTTCCCCAACCACGCCGAGAAGCAGAAGCAGTTCGAGT TTGTGGGCTCCGCCCCCACCCGCCGGACCAAGCGCACACGGCGGCCCCAGCCCCTCACG GGATCGGGATCGGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCCG GGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGG ACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGT TCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGA GCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGC TGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGA GAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCC CCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTT CTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTAC AAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCAC CGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGA GGCTCTGCACAACCACTACACGCAGAAaAGCCTCTCCCTGTCTCCGGGTAAA 24 FGF- Human FGF-17 AA204-ACTCAGGGGGAGAATCACCCGTCTCCTAA 17d204- 216 deletion mutant-TTTTAACCAGTACGTGAGGGACCAGGGCG 216- linker1- hFcmCCATGACCGACCAGCTGAGCAGGCGGCAG linker1- nucleotide sequenceATCCGCGAGTACCAACTCTACAGCAGGAC hFcm CAGTGGCAAGCACGTGCAGGTCACCGGGCGTCGCATCTCCGCCACCGCCGAGGACGGC AACAAGTTTGCCAAGCTCATAGTGGAGACGGACACGTTTGGCAGCCGGGTTCGCATCA AAGGGGCTGAGAGTGAGAAGTACATCTGTATGAACAAGAGGGGCAAGCTCATCGGGAA GCCCAGCGGGAAGAGCAAAGACTGCGTGTTCACGGAGATCGTGCTGGAGAACAACTAT ACGGCCTTCCAGAACGCCCGGCACGAGGGCTGGTTCATGGCCTTCACGCGGCAGGGGC GGCCCCGCCAGGCTTCCCGCAGCCGCCAGAACCAGCGCGAGGCCCACTTCATCAAGCG CCTCTACCAAGGCCAGCTGCCCTTCCCCAACCACGCCGAGAAGCAGAAGCAGTTCGAGT TTGTGGGCTCCGCCCCCACCGGATCGGGATCGGACAAAACTCACACATGCCCACCGTGC CCAGCACCTGAAGCTGCCGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGA CACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACG AAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAA GACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCG TCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGC CCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCAC AGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACC TGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCA GCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCC TCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATG CTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAaAGCCTCTCCCTGTCTCC GGGTAAA 25 FGF- Human FGF-17 AA181-ACTCAGGGGGAGAATCACCCGTCTCCTAA 17d181- 216 deletion mutant-TTTTAACCAGTACGTGAGGGACCAGGGCG 216- linker 1- hFcmCCATGACCGACCAGCTGAGCAGGCGGCAG linker1- nucleotide sequenceATCCGCGAGTACCAACTCTACAGCAGGAC hFcm CAGTGGCAAGCACGTGCAGGTCACCGGGCGTCGCATCTCCGCCACCGCCGAGGACGGC AACAAGTTTGCCAAGCTCATAGTGGAGACGGACACGTTTGGCAGCCGGGTTCGCATCA AAGGGGCTGAGAGTGAGAAGTACATCTGTATGAACAAGAGGGGCAAGCTCATCGGGAA GCCCAGCGGGAAGAGCAAAGACTGCGTGTTCACGGAGATCGTGCTGGAGAACAACTAT ACGGCCTTCCAGAACGCCCGGCACGAGGGCTGGTTCATGGCCTTCACGCGGCAGGGGC GGCCCCGCCAGGCTTCCCGCAGCCGCCAGAACCAGCGCGAGGCCCACTTCATCAAGCG CCTCTACCAAGGATCGGGATCGGACAAAACTCACACATGCCCACCGTGCCCAGCACCTG AAGCTGCCGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATG ATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGA GGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCG CGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCA GGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCC CCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACA CCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTC AAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGA ACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGC AAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGAT GCATGAGGCTCTGCACAACCACTACACGCAGAAaAGCCTCTCCCTGTCTCCGGGTAAA 26 FGF-17 HumanFGF-17ACTCAGGGGGAGAATCACCCGTCTCCTAA R204Q R204Q K207Q mutant-TTTTAACCAGTACGTGAGGGACCAGGGCG K207Q- linker1-hFcm CCATGACCGACCAGCTGAGCAGGCGGCAG linker1- nucleotide sequenceATCCGCGAGTACCAACTCTACAGCAGGAC hFcm CAGTGGCAAGCACGTGCAGGTCACCGGGCGTCGCATCTCCGCCACCGCCGAGGACGGC AACAAGTTTGCCAAGCTCATAGTGGAGACGGACACGTTTGGCAGCCGGGTTCGCATCA AAGGGGCTGAGAGTGAGAAGTACATCTGTATGAACAAGAGGGGCAAGCTCATCGGGAA GCCCAGCGGGAAGAGCAAAGACTGCGTGTTCACGGAGATCGTGCTGGAGAACAACTAT ACGGCCTTCCAGAACGCCCGGCACGAGGGCTGGTTCATGGCCTTCACGCGGCAGGGGC GGCCCCGCCAGGCTTCCCGCAGCCGCCAGAACCAGCGCGAGGCCCACTTCATCAAGCG CCTCTACCAAGGCCAGCTGCCCTTCCCCAACCACGCCGAGAAGCAGAAGCAGTTCGAGT TTGTGGGCTCCGCCCCCACCCaGCGGACCcAGCGCACACGGCGGCCCCAGCCCCTCACG GGATCGGGATCGGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCCG GGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGG ACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGT TCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGA GCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGC TGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGA GAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCC CCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTT CTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTAC AAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCAC CGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGG CTCTGCACAACCACTACACGCAGAAaAGCCTCTCCCTGTCTCCGGGTAAA 27 FGF- Human FGF17 AA197-ACTCAGGGGGAGAATCACCCGTCTCCTAA 17d197- 216 deletion mutant-TTTTAACCAGTACGTGAGGGACCAGGGCG 216- linker1- hFcmCCATGACCGACCAGCTGAGCAGGCGGCAG linker1- nucleotide sequenceATCCGCGAGTACCAACTCTACAGCAGGAC hFcm CAGTGGCAAGCACGTGCAGGTCACCGGGCGTCGCATCTCCGCCACCGCCGAGGACGGC AACAAGTTTGCCAAGCTCATAGTGGAGACGGACACGTTTGGCAGCCGGGTTCGCATCA AAGGGGCTGAGAGTGAGAAGTACATCTGTATGAACAAGAGGGGCAAGCTCATCGGGAA GCCCAGCGGGAAGAGCAAAGACTGCGTGTTCACGGAGATCGTGCTGGAGAACAACTAT ACGGCCTTCCAGAACGCCCGGCACGAGGGCTGGTTCATGGCCTTCACGCGGCAGGGGC GGCCCCGCCAGGCTTCCCGCAGCCGCCAGAACCAGCGCGAGGCCCACTTCATCAAGCG CCTCTACCAAGGCCAGCTGCCCTTCCCCAACCACGCCGAGAAGCAGAAGCAGTTCGAGG GATCGGGATCGGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCCGG GGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTT CAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAG CAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCT GAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAG AAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCC CATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTC TATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACA AGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACC GTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGG CTCTGCACAACCACTACACGCAGAAaAGCCTCTCCCTGTCTCCGGGTAAA 28 FGF-17 Human FGF-17 K191AACTCAGGGGGAGAATCACCCGTCTCCTAA K191A K193A K193A S200A mutant-TTTTAACCAGTACGTGAGGGACCAGGGCG S200A- linker1- hFcmCCATGACCGACCAGCTGAGCAGGCGGCAG linker1- nucleotide sequenceATCCGCGAGTACCAACTCTACAGCAGGAC hFcm CAGTGGCAAGCACGTGCAGGTCACCGGGCGTCGCATCTCCGCCACCGCCGAGGACGGC AACAAGTTTGCCAAGCTCATAGTGGAGACGGACACGTTTGGCAGCCGGGTTCGCATCA AAGGGGCTGAGAGTGAGAAGTACATCTGTATGAACAAGAGGGGCAAGCTCATCGGGAA GCCCAGCGGGAAGAGCAAAGACTGCGTGTTCACGGAGATCGTGCTGGAGAACAACTAT ACGGCCTTCCAGAACGCCCGGCACGAGGGCTGGTTCATGGCCTTCACGCGGCAGGGGC GGCCCCGCCAGGCTTCCCGCAGCCGCCAGAACCAGCGCGAGGCCCACTTCATCAAGCG CCTCTACCAAGGCCAGCTGCCCTTCCCCAACCACGCCGAGGctCAGGcaCAGTTCGAGTTT GTGGGCgCtGCCCCCACCCGCCGGACCAAGCGCACACGGCGGCCCCAGCCCCTCACGGG ATCGGGATCGGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCCGGG GGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACC CCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCA ACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCA GTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGA ATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAA AACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCA TCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTA TCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAG ACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTG GACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCT GCACAACCACTACACGCAGAAaAGCCTCTCCCTGTCTCCGGGTAAA 29 FGF-17- Full length of humanACTCAGGGGGAGAATCACCCGTCTCCTAA linker2- FGF17 linker2- hFcmTTTTAACCAGTACGTGAGGGACCAGGGCG hFcm nucleotide sequenceCCATGACCGACCAGCTGAGCAGGCGGCAG ATCCGCGAGTACCAACTCTACAGCAGGACCAGTGGCAAGCACGTGCAGGTCACCGGGC GTCGCATCTCCGCCACCGCCGAGGACGGCAACAAGTTTGCCAAGCTCATAGTGGAGAC GGACACGTTTGGCAGCCGGGTTCGCATCAAAGGGGCTGAGAGTGAGAAGTACATCTGT ATGAACAAGAGGGGCAAGCTCATCGGGAAGCCCAGCGGGAAGAGCAAAGACTGCGTGT TCACGGAGATCGTGCTGGAGAACAACTATACGGCCTTCCAGAACGCCCGGCACGAGGG CTGGTTCATGGCCTTCACGCGGCAGGGGCGGCCCCGCCAGGCTTCCCGCAGCCGCCAG AACCAGCGCGAGGCCCACTTCATCAAGCGCCTCTACCAAGGCCAGCTGCCCTTCCCCAA CCACGCCGAGAAGCAGAAGCAGTTCGAGTTTGTGGGCTCCGCCCCCACCCGCCGGACCA AGCGCACACGGCGGCCCCAGCCCCTCACGGGATCTGGGAGCGCTGACAAAACTCACAC ATGCCCACCGTGCCCAGCACCTGAAGCTGCCGGGGGACCGTCAGTCTTCCTCTTCCCCC CAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGT GGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAG GTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGG TCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAG GTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCA GCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAAC CAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTG GGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCC GACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGG GAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAaA GCCTCTCCCTGTCTCCGGGTAAA 30 FGF-Human FGF17 AA204- ACTCAGGGGGAGAATCACCCGTCTCCTAA 17d204-216 deletion mutant TTTTAACCAGTACGTGAGGGACCAGGGCG 216linker2-linker2- hFcm CCATGACCGACCAGCTGAGCAGGCGGCAG hFcm nucleotide sequenceATCCGCGAGTACCAACTCTACAGCAGGAC CAGTGGCAAGCACGTGCAGGTCACCGGGCGTCGCATCTCCGCCACCGCCGAGGACGGC AACAAGTTTGCCAAGCTCATAGTGGAGACGGACACGTTTGGCAGCCGGGTTCGCATCA AAGGGGCTGAGAGTGAGAAGTACATCTGTATGAACAAGAGGGGCAAGCTCATCGGGAA GCCCAGCGGGAAGAGCAAAGACTGCGTGTTCACGGAGATCGTGCTGGAGAACAACTAT ACGGCCTTCCAGAACGCCCGGCACGAGGGCTGGTTCATGGCCTTCACGCGGCAGGGGC GGCCCCGCCAGGCTTCCCGCAGCCGCCAGAACCAGCGCGAGGCCCACTTCATCAAGCG CCTCTACCAAGGCCAGCTGCCCTTCCCCAACCACGCCGAGAAGCAGAAGCAGTTCGAGT TTGTGGGCTCCGCCCCCACCGGATCTGGGAGCGCTGACAAAACTCACACATGCCCACCG TGCCCAGCACCTGAAGCTGCCGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAA GGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCC ACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGC CAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCA CCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAA AGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAA CCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCT GACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATG GGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTC TTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTC ATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAaAGCCTCTCCCTGTC TCCGGGTAAA 31 FGF- Human FGF17 AA181-ACTCAGGGGGAGAATCACCCGTCTCCTAA 17d181- 216 deletion mutantTTTTAACCAGTACGTGAGGGACCAGGGCG 216linker2- linker2- hFcmCCATGACCGACCAGCTGAGCAGGCGGCAG hFcm nucleotide sequenceATCCGCGAGTACCAACTCTACAGCAGGAC CAGTGGCAAGCACGTGCAGGTCACCGGGCGTCGCATCTCCGCCACCGCCGAGGACGGC AACAAGTTTGCCAAGCTCATAGTGGAGACGGACACGTTTGGCAGCCGGGTTCGCATCA AAGGGGCTGAGAGTGAGAAGTACATCTGTATGAACAAGAGGGGCAAGCTCATCGGGAA GCCCAGCGGGAAGAGCAAAGACTGCGTGTTCACGGAGATCGTGCTGGAGAACAACTAT ACGGCCTTCCAGAACGCCCGGCACGAGGGCTGGTTCATGGCCTTCACGCGGCAGGGGC GGCCCCGCCAGGCTTCCCGCAGCCGCCAGAACCAGCGCGAGGCCCACTTCATCAAGCG CCTCTACCAAGGATCTGGGAGCGCTGACAAAACTCACACATGCCCACCGTGCCCAGCA CCTGAAGCTGCCGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTC ATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCC TGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAG CCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCA CCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGC GCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGT ACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCT GGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGG AGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTAC AGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGT GATGCATGAGGCTCTGCACAACCACTACACGCAGAAaAGCCTCTCCCTGTCTCCGGGTA AA 32 FGF- Human FGF-17 R204QACTCAGGGGGAGAATCACCCGTCTCCTAA 17R204Q K207Q mutant linker2-TTTTAACCAGTACGTGAGGGACCAGGGCG K207Q hFcm nucleotideCCATGACCGACCAGCTGAGCAGGCGGCAG linker2- sequenceATCCGCGAGTACCAACTCTACAGCAGGAC hFcm CAGTGGCAAGCACGTGCAGGTCACCGGGCGTCGCATCTCCGCCACCGCCGAGGACGGC AACAAGTTTGCCAAGCTCATAGTGGAGACGGACACGTTTGGCAGCCGGGTTCGCATCA AAGGGGCTGAGAGTGAGAAGTACATCTGTATGAACAAGAGGGGCAAGCTCATCGGGAA GCCCAGCGGGAAGAGCAAAGACTGCGTGTTCACGGAGATCGTGCTGGAGAACAACTAT ACGGCCTTCCAGAACGCCCGGCACGAGGGCTGGTTCATGGCCTTCACGCGGCAGGGGC GGCCCCGCCAGGCTTCCCGCAGCCGCCAGAACCAGCGCGAGGCCCACTTCATCAAGCG CCTCTACCAAGGCCAGCTGCCCTTCCCCAACCACGCCGAGAAGCAGAAGCAGTTCGAGT TTGTGGGCTCCGCCCCCACCCaGCGGACCcAGCGCACACGGCGGCCCCAGCCCCTCACG GGATCTGGGAGCGCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTG CCGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCC CGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCA AGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGA GGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACT GGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATC GAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGC CCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGG CTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACT ACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTC ACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGA GGCTCTGCACAACCACTACACGCAGAAaAGCCTCTCCCTGTCTCCGGGTAAA 33 FGF- Human FGF17 AA197-ACTCAGGGGGAGAATCACCCGTCTCCTAA 17d197- 216 deletion mutantTTTTAACCAGTACGTGAGGGACCAGGGCG 216linker2- linker2- hFcmCCATGACCGACCAGCTGAGCAGGCGGCAG hFcm nucleotide sequenceATCCGCGAGTACCAACTCTACAGCAGGAC CAGTGGCAAGCACGTGCAGGTCACCGGGCGTCGCATCTCCGCCACCGCCGAGGACGGC AACAAGTTTGCCAAGCTCATAGTGGAGACGGACACGTTTGGCAGCCGGGTTCGCATCA AAGGGGCTGAGAGTGAGAAGTACATCTGTATGAACAAGAGGGGCAAGCTCATCGGGAA GCCCAGCGGGAAGAGCAAAGACTGCGTGTTCACGGAGATCGTGCTGGAGAACAACTAT ACGGCCTTCCAGAACGCCCGGCACGAGGGCTGGTTCATGGCCTTCACGCGGCAGGGGC GGCCCCGCCAGGCTTCCCGCAGCCGCCAGAACCAGCGCGAGGCCCACTTCATCAAGCG CCTCTACCAAGGCCAGCTGCCCTTCCCCAACCACGCCGAGAAGCAGAAGCAGTTCGAGG GATCTGGGAGCGCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCC GGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCG GACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAG TTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGG AGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGG CTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCG AGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCC CCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGC TTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTA CAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCA CCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAG GCTCTGCACAACCACTACACGCAGAAaAGCCTCTCCCTGTCTCCGGGTAAA 34 FGF-17 Human FGF-17 K191AACTCAGGGGGAGAATCACCCGTCTCCTAA K191A K193A S200A mutantTTTTAACCAGTACGTGAGGGACCAGGGCG K193A S200A linker2- hFcmCCATGACCGACCAGCTGAGCAGGCGGCAG linker2- nucleotide sequenceATCCGCGAGTACCAACTCTACAGCAGGAC hFcm CAGTGGCAAGCACGTGCAGGTCACCGGGCGTCGCATCTCCGCCACCGCCGAGGACGGC AACAAGTTTGCCAAGCTCATAGTGGAGACGGACACGTTTGGCAGCCGGGTTCGCATCA AAGGGGCTGAGAGTGAGAAGTACATCTGTATGAACAAGAGGGGCAAGCTCATCGGGAA GCCCAGCGGGAAGAGCAAAGACTGCGTGTTCACGGAGATCGTGCTGGAGAACAACTAT ACGGCCTTCCAGAACGCCCGGCACGAGGGCTGGTTCATGGCCTTCACGCGGCAGGGGC GGCCCCGCCAGGCTTCCCGCAGCCGCCAGAACCAGCGCGAGGCCCACTTCATCAAGCG CCTCTACCAAGGCCAGCTGCCCTTCCCCAACCACGCCGAGGCTCAGGCACAGTTCGAGT TTGTGGGCGCTGCCCCCACCCGCCGGACCAAGCGCACACGGCGGCCCCAGCCCCTCACG GGATCTGGGAGCGCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTG CCGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCC CGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCA AGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGA GGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACT GGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATC GAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGC CCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGG CTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACT ACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTC ACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGA GGCTCTGCACAACCACTACACGCAGAAaAGCCTCTCCCTGTCTCCGGGTAAA 35 6xHis- His tagged HSA fusionCACCATCACCATCACCATAGCGGCGATGC HSA- FGF17 with a longACACAAGAGTGAGGTTGCTCATCGGTTTA linker3- linker nucleotideAAGATTTGGGAGAAGAAAATTTCAAAGCC FGF17 sequenceTTGGTGTTGATTGCCTTTGCTCAGTATCTTC AGCAGTGTCCATTTGAAGATCATGTAAAATTAGTGAATGAAGTAACTGAATTTGCAAAA ACATGTGTTGCTGATGAGTCAGCTGAAAATTGTGACAAATCACTTCATACCCTTTTTGGA GACAAATTATGCACAGTTGCAACTCTTCGTGAAACCTATGGTGAAATGGCTGACTGCTG TGCAAAACAAGAACCTGAGAGAAATGAATGCTTCTTGCAACACAAAGATGACAACCCA AACCTCCCCCGATTGGTGAGACCAGAGGTTGATGTGATGTGCACTGCTTTTCATGACAA TGAAGAGACATTTTTGAAAAAATACTTATATGAAATTGCCAGAAGACATCCTTACTTTTA TGCCCCGGAACTCCTTTTCTTTGCTAAAAGGTATAAAGCTGCTTTTACAGAATGTTGCCA AGCTGCTGATAAAGCTGCCTGCCTGTTGCCAAAGCTCGATGAACTTCGGGATGAAGGGA AGGCTTCGTCTGCCAAACAGAGACTCAAGTGTGCCAGTCTCCAAAAATTTGGAGAAAG AGCTTTCAAAGCATGGGCAGTAGCTCGCCTGAGCCAGAGATTTCCCAAAGCTGAGTTTG CAGAAGTTTCCAAGTTAGTGACAGATCTTACCAAAGTCCACACGGAATGCTGCCATGGA GATCTGCTTGAATGTGCTGATGACAGGGCGGACCTTGCCAAGTATATCTGTGAAAATCA AGATTCGATCTCCAGTAAACTGAAGGAATGCTGTGAAAAACCTCTGTTGGAAAAATCC CACTGCATTGCCGAAGTGGAAAATGATGAGATGCCTGCTGACTTGCCTTCATTAGCTGC TGATTTTGTTGAAAGTAAGGATGTTTGCAAAAACTATGCTGAGGCAAAGGATGTCTTCCT GGGCATGTTTTTGTATGAATATGCAAGAAGGCATCCTGATTACTCTGTCGTGCTGCTGCT GAGACTTGCCAAGACATATGAAACCACTCTAGAGAAGTGCTGTGCCGCTGCAGATCCTC ATGAATGCTATGCCAAAGTGTTCGATGAATTTAAACCTCTTGTGGAAGAGCCTCAGAATT TAATCAAACAAAATTGTGAGCTTTTTGAGCAGCTTGGAGAGTACAAATTCCAGAATGCG CTATTAGTTCGTTACACCAAGAAAGTACCCCAAGTGTCAACTCCAACTCTTGTAGAGGTC TCAAGAAACCTAGGAAAAGTGGGCAGCAAATGTTGTAAACATCCTGAAGCAAAAAGAA TGCCCTGTGCAGAAGACTATCTATCCGTGGTCCTGAACCAGTTATGTGTGTTGCATGAGA AAACGCCAGTAAGTGACAGAGTCACCAAATGCTGCACAGAATCCTTGGTGAACAGGCG ACCATGCTTTTCAGCTCTGGAAGTCGATGAAACATACGTTCCCAAAGAGTTTAATGCTGA AACATTCACCTTCCATGCAGATATATGCACACTTTCTGAGAAGGAGAGACAAATCAAGA AACAAACTGCACTTGTTGAGCTCGTGAAACACAAGCCCAAGGCAACAAAAGAGCAACT GAAAGCTGTTATGGATGATTTCGCAGCTTTTGTAGAGAAGTGCTGCAAGGCTGACGATA AGGAGACCTGCTTTGCCGAGGAGGGTAAAAAACTTGTTGCTGCAAGTCAAGCTGCCTTA GGCTTAGGCGGAGGCGGTAGCGGAGGCGGTGGCTCCGGTGGCGGAGGGTCTACTCAGG GGGAGAATCACCCGTCTCCTAATTTTAACCAGTACGTGAGGGACCAGGGCGCCATGACC GACCAGCTGAGCAGGCGGCAGATCCGCGAGTACCAACTCTACAGCAGGACCAGTGGCA AGCACGTGCAGGTCACCGGGCGTCGCATCTCCGCCACCGCCGAGGACGGCAACAAGTT TGCCAAGCTCATAGTGGAGACGGACACGTTTGGCAGCCGGGTTCGCATCAAAGGGGCT GAGAGTGAGAAGTACATCTGTATGAACAAGAGGGGCAAGCTCATCGGGAAGCCCAGCG GGAAGAGCAAAGACTGCGTGTTCACGGAGATCGTGCTGGAGAACAACTATACGGCCTTC CAGAACGCCCGGCACGAGGGCTGGTTCATGGCCTTCACGCGGCAGGGGCGGCCCCGCC AGGCTTCCCGCAGCCGCCAGAACCAGCGCGAGGCCCACTTCATCAAGCGCCTCTACCAA GGCCAGCTGCCCTTCCCCAACCACGCCGAGAAGCAGAAGCAGTTCGAGTTTGTGGGCT CCGCCCCCACCCGCCGGACCAAGCGCACACGGCGGCCCCAGCCCCTCACG 36 FGF17- Human FGF17- linker3-ACTCAGGGGGAGAATCACCCGTCTCCTAA linker3- hFc4 nucleotideTTTTAACCAGTACGTGAGGGACCAGGGCG hFc4 sequenceCCATGACCGACCAGCTGAGCAGGCGGCAG ATCCGCGAGTACCAACTCTACAGCAGGACCAGTGGCAAGCACGTGCAGGTCACCGGGC GTCGCATCTCCGCCACCGCCGAGGACGGCAACAAGTTTGCCAAGCTCATAGTGGAGAC GGACACGTTTGGCAGCCGGGTTCGCATCAAAGGGGCTGAGAGTGAGAAGTACATCTGT ATGAACAAGAGGGGCAAGCTCATCGGGAAGCCCAGCGGGAAGAGCAAAGACTGCGTGT TCACGGAGATCGTGCTGGAGAACAACTATACGGCCTTCCAGAACGCCCGGCACGAGGG CTGGTTCATGGCCTTCACGCGGCAGGGGCGGCCCCGCCAGGCTTCCCGCAGCCGCCAG AACCAGCGCGAGGCCCACTTCATCAAGCGCCTCTACCAAGGCCAGCTGCCCTTCCCCAA CCACGCCGAGAAGCAGAAGCAGTTCGAGTTTGTGGGCTCCGCCCCCACCCGCCGGACCA AGCGCACACGGCGGCCCCAGCCCCTCACGGGCGGAGGCGGTAGCGGAGGCGGTGGCTC CGGTGGCGGAGGGTCTGAGTCCAAATATGGTCCCCCATGCCCACCCTGCCCAGCACCTG AGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATG ATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGA GGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGC GGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAG GACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTC CATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACC CTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCA AAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGA ACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGC AGGCTCACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGAT GCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAA 37 FGF17- Human FGF17- linker3-ACTCAGGGGGAGAATCACCCGTCTCCTAA hFc4 hFc4 nucleotideTTTTAACCAGTACGTGAGGGACCAGGGCG sequence CCATGACCGACCAGCTGAGCAGGCGGCAGATCCGCGAGTACCAACTCTACAGCAGGAC CAGTGGCAAGCACGTGCAGGTCACCGGGCGTCGCATCTCCGCCACCGCCGAGGACGGC AACAAGTTTGCCAAGCTCATAGTGGAGACGGACACGTTTGGCAGCCGGGTTCGCATCA AAGGGGCTGAGAGTGAGAAGTACATCTGTATGAACAAGAGGGGCAAGCTCATCGGGAA GCCCAGCGGGAAGAGCAAAGACTGCGTGTTCACGGAGATCGTGCTGGAGAACAACTAT ACGGCCTTCCAGAACGCCCGGCACGAGGGCTGGTTCATGGCCTTCACGCGGCAGGGGC GGCCCCGCCAGGCTTCCCGCAGCCGCCAGAACCAGCGCGAGGCCCACTTCATCAAGCG CCTCTACCAAGGCCAGCTGCCCTTCCCCAACCACGCCGAGAAGCAGAAGCAGTTCGAGT TTGTGGGCTCCGCCCCCACCCGCCGGACCAAGCGCACACGGCGGCCCCAGCCCCTCACG GAGTCCAAATATGGTCCCCCATGCCCACCCTGCCCAGCACCTGAGTTCCTGGGGGGACC ATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGA GGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGG TACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCA ACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGC AAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCAT CTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGG AGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGC GACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGC CTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTCACCGTGGACAAG AGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAA CCACTACACACAGAAGAGCCTCTCCCTGTC TCTGGGTAAA38 hFc4L- hFc4- linker3- human GAGTCCAAATATGGTCCCCCATGCCCACCC FGF17FGF17 Nucleotide TGCCCAGCACCTGAGTTCCTGGGGGGACC sequenceATCAGTCTTCCTGTTCCCCCCAAAACCCAA GGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCC AGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGC CAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCA CCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAA AGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGC CACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTG ACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGG GCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCT TCCTCTACAGCAGGCTCACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTC ATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTC TCTGGGTAAAGGCGGAGGCGGTAGCGGAGGCGGTGGCTCCGGTGGCGGAGGGTCTACT CAGGGGGAGAATCACCCGTCTCCTAATTTTAACCAGTACGTGAGGGACCAGGGCGCCAT GACCGACCAGCTGAGCAGGCGGCAGATCCGCGAGTACCAACTCTACAGCAGGACCAGT GGCAAGCACGTGCAGGTCACCGGGCGTCGCATCTCCGCCACCGCCGAGGACGGCAACA AGTTTGCCAAGCTCATAGTGGAGACGGACACGTTTGGCAGCCGGGTTCGCATCAAAGG GGCTGAGAGTGAGAAGTACATCTGTATGAACAAGAGGGGCAAGCTCATCGGGAAGCCC AGCGGGAAGAGCAAAGACTGCGTGTTCACGGAGATCGTGCTGGAGAACAACTATACGG CCTTCCAGAACGCCCGGCACGAGGGCTGGTTCATGGCCTTCACGCGGCAGGGGCGGCC CCGCCAGGCTTCCCGCAGCCGCCAGAACCAGCGCGAGGCCCACTTCATCAAGCGCCTCT ACCAAGGCCAGCTGCCCTTCCCCAACCACGCCGAGAAGCAGAAGCAGTTCGAGTTTGT GGGCTCCGCCCCCACCCGCCGGACCAAGCGCACACGGCGGCCCCAGCCCCTCACG 39 IGF2 nucleotide sequenceGCTTACCGCCCCAGTGAGACCCTGTGCGGC GGGGAGCTGGTGGACACCCTCCAGTTCGTCTGTGGGGACCGCGGCTTCTACTTCAGCAG GCCCGCAAGCCGTGTGAGCCGTCGCAGCCGTGGCATCGTTGAGGAGTGCTGTTTCCGCA GCTGTGACCTGGCCCTCCTGGAGACGTACTGTGCTACCCCCGCCAAGTCCGAG 40 IGF2- nucleotide sequenceGCTTACCGCCCCAGTGAGACCCTGTGCGGC linker1- GGGGAGCTGGTGGACACCCTCCAGTTCGThFcm CTGTGGGGACCGCGGCTTCTACTTCAGCAG GCCCGCAAGCCGTGTGAGCCGTCGCAGCCGTGGCATCGTTGAGGAGTGCTGTTTCCGCA GCTGTGACCTGGCCCTCCTGGAGACGTACTGTGCTACCCCCGCCAAGTCCGAGGGATCG GGATCGGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCCGGGGGAC CGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTG AGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTG GTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTAC AACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGG CAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACC ATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCG GGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCA GCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCAC GCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAA GAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACA ACCACTACACGCAGAAaAGCCTCTCCCTGTCTCCGGGTAAA 41 IGF2- nucleotide sequence GCTTACCGCCCCAGTGAGACCCTGTGCGGClinker2- GGGGAGCTGGTGGACACCCTCCAGTTCGT hFcmCTGTGGGGACCGCGGCTTCTACTTCAGCAG GCCCGCAAGCCGTGTGAGCCGTCGCAGCCGTGGCATCGTTGAGGAGTGCTGTTTCCGCA GCTGTGACCTGGCCCTCCTGGAGACGTACTGTGCTACCCCCGCCAAGTCCGAGGGATCT GGGAGCGCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCCGGGG GACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCC CTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAA CTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAG TACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAA TGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAA ACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT CCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTAT CCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGA CCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGG ACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTG CACAACCACTACACGCAGAAaAGCCTCTCCCTGTCTCCGGGTAAA 42 6xHis- His tagged HSA fusionCACCATCACCATCACCATAGCGGCGATGC HSA- IGF2 with a long linkerACACAAGAGTGAGGTTGCTCATCGGTTTA linker3- nucleotide sequenceAAGATTTGGGAGAAGAAAATTTCAAAGCC IGF2 TTGGTGTTGATTGCCTTTGCTCAGTATCTTCAGCAGTGTCCATTTGAAGATCATGTAAAAT TAGTGAATGAAGTAACTGAATTTGCAAAAACATGTGTTGCTGATGAGTCAGCTGAAAAT TGTGACAAATCACTTCATACCCTTTTTGGAGACAAATTATGCACAGTTGCAACTCTTCGT GAAACCTATGGTGAAATGGCTGACTGCTGTGCAAAACAAGAACCTGAGAGAAATGAAT GCTTCTTGCAACACAAAGATGACAACCCAAACCTCCCCCGATTGGTGAGACCAGAGGT TGATGTGATGTGCACTGCTTTTCATGACAATGAAGAGACATTTTTGAAAAAATACTTATA TGAAATTGCCAGAAGACATCCTTACTTTTATGCCCCGGAACTCCTTTTCTTTGCTAAAAG GTATAAAGCTGCTTTTACAGAATGTTGCCAAGCTGCTGATAAAGCTGCCTGCCTGTTGCC AAAGCTCGATGAACTTCGGGATGAAGGGAAGGCTTCGTCTGCCAAACAGAGACTCAAG TGTGCCAGTCTCCAAAAATTTGGAGAAAGAGCTTTCAAAGCATGGGCAGTAGCTCGCCT GAGCCAGAGATTTCCCAAAGCTGAGTTTGCAGAAGTTTCCAAGTTAGTGACAGATCTTA CCAAAGTCCACACGGAATGCTGCCATGGAGATCTGCTTGAATGTGCTGATGACAGGGC GGACCTTGCCAAGTATATCTGTGAAAATCAAGATTCGATCTCCAGTAAACTGAAGGAAT GCTGTGAAAAACCTCTGTTGGAAAAATCCCACTGCATTGCCGAAGTGGAAAATGATGA GATGCCTGCTGACTTGCCTTCATTAGCTGCTGATTTTGTTGAAAGTAAGGATGTTTGCAA AAACTATGCTGAGGCAAAGGATGTCTTCCTGGGCATGTTTTTGTATGAATATGCAAGAAG GCATCCTGATTACTCTGTCGTGCTGCTGCTGAGACTTGCCAAGACATATGAAACCACTC TAGAGAAGTGCTGTGCCGCTGCAGATCCTCATGAATGCTATGCCAAAGTGTTCGATGAAT TTAAACCTCTTGTGGAAGAGCCTCAGAATTTAATCAAACAAAATTGTGAGCTTTTTGAGC AGCTTGGAGAGTACAAATTCCAGAATGCGCTATTAGTTCGTTACACCAAGAAAGTACCC CAAGTGTCAACTCCAACTCTTGTAGAGGTCTCAAGAAACCTAGGAAAAGTGGGCAGCAA ATGTTGTAAACATCCTGAAGCAAAAAGAATGCCCTGTGCAGAAGACTATCTATCCGTGG TCCTGAACCAGTTATGTGTGTTGCATGAGAAAACGCCAGTAAGTGACAGAGTCACCAAA TGCTGCACAGAATCCTTGGTGAACAGGCGACCATGCTTTTCAGCTCTGGAAGTCGATGA AACATACGTTCCCAAAGAGTTTAATGCTGAAACATTCACCTTCCATGCAGATATATGCAC ACTTTCTGAGAAGGAGAGACAAATCAAGAAACAAACTGCACTTGTTGAGCTCGTGAAA CACAAGCCCAAGGCAACAAAAGAGCAACTGAAAGCTGTTATGGATGATTTCGCAGCTTT TGTAGAGAAGTGCTGCAAGGCTGACGATAAGGAGACCTGCTTTGCCGAGGAGGGTAAA AAACTTGTTGCTGCAAGTCAAGCTGCCTTAGGCTTAGGCGGAGGCGGTAGCGGAGGCGG TGGCTCCGGTGGCGGAGGGTCTGCTTACCGCCCCAGTGAGACCCTGTGCGGCGGGGAGC TGGTGGACACCCTCCAGTTCGTCTGTGGGGACCGCGGCTTCTACTTCAGCAGGCCCGCAA GCCGTGTGAGCCGTCGCAGCCGTGGCATCGTTGAGGAGTGCTGTTTCCGCAGCTGTGAC CTGGCCCTCCTGGAGACGTACTGTGCTACCCCCGCCAAGTCCGAG 43 6xHis- His tagged HSA fusionCACCATCACCATCACCATAGCGGCGATGC HSA- IGF2 R61A mutant withACACAAGAGTGAGGTTGCTCATCGGTTTA linker3- a long linker AAGATTTGGGAGAAGAAAATTTCAAAGCC IGF2R61A nucleotide sequenceTTGGTGTTGATTGCCTTTGCTCAGTATCTTC AGCAGTGTCCATTTGAAGATCATGTAAAATTAGTGAATGAAGTAACTGAATTTGCAAAA ACATGTGTTGCTGATGAGTCAGCTGAAAATTGTGACAAATCACTTCATACCCTTTTTGGA GACAAATTATGCACAGTTGCAACTCTTCGTGAAACCTATGGTGAAATGGCTGACTGCTG TGCAAAACAAGAACCTGAGAGAAATGAATGCTTCTTGCAACACAAAGATGACAACCCA AACCTCCCCCGATTGGTGAGACCAGAGGTTGATGTGATGTGCACTGCTTTTCATGACAA TGAAGAGACATTTTTGAAAAAATACTTATATGAAATTGCCAGAAGACATCCTTACTTTTA TGCCCCGGAACTCCTTTTCTTTGCTAAAAGGTATAAAGCTGCTTTTACAGAATGTTGCCA AGCTGCTGATAAAGCTGCCTGCCTGTTGCCAAAGCTCGATGAACTTCGGGATGAAGGGA AGGCTTCGTCTGCCAAACAGAGACTCAAGTGTGCCAGTCTCCAAAAATTTGGAGAAAG AGCTTTCAAAGCATGGGCAGTAGCTCGCCTGAGCCAGAGATTTCCCAAAGCTGAGTTTG CAGAAGTTTCCAAGTTAGTGACAGATCTTACCAAAGTCCACACGGAATGCTGCCATGGA GATCTGCTTGAATGTGCTGATGACAGGGCGGACCTTGCCAAGTATATCTGTGAAAATCA AGATTCGATCTCCAGTAAACTGAAGGAATGCTGTGAAAAACCTCTGTTGGAAAAATCC CACTGCATTGCCGAAGTGGAAAATGATGAGATGCCTGCTGACTTGCCTTCATTAGCTGC TGATTTTGTTGAAAGTAAGGATGTTTGCAAAAACTATGCTGAGGCAAAGGATGTCTTCCT GGGCATGTTTTTGTATGAATATGCAAGAAGGCATCCTGATTACTCTGTCGTGCTGCTGCT GAGACTTGCCAAGACATATGAAACCACTCTAGAGAAGTGCTGTGCCGCTGCAGATCCTC ATGAATGCTATGCCAAAGTGTTCGATGAATTTAAACCTCTTGTGGAAGAGCCTCAGAATT TAATCAAACAAAATTGTGAGCTTTTTGAGCAGCTTGGAGAGTACAAATTCCAGAATGCG CTATTAGTTCGTTACACCAAGAAAGTACCCCAAGTGTCAACTCCAACTCTTGTAGAGGTC TCAAGAAACCTAGGAAAAGTGGGCAGCAAATGTTGTAAACATCCTGAAGCAAAAAGAA TGCCCTGTGCAGAAGACTATCTATCCGTGGTCCTGAACCAGTTATGTGTGTTGCATGAGA AAACGCCAGTAAGTGACAGAGTCACCAAATGCTGCACAGAATCCTTGGTGAACAGGCG ACCATGCTTTTCAGCTCTGGAAGTCGATGAAACATACGTTCCCAAAGAGTTTAATGCTGA AACATTCACCTTCCATGCAGATATATGCACACTTTCTGAGAAGGAGAGACAAATCAAGA AACAAACTGCACTTGTTGAGCTCGTGAAACACAAGCCCAAGGCAACAAAAGAGCAACT GAAAGCTGTTATGGATGATTTCGCAGCTTTTGTAGAGAAGTGCTGCAAGGCTGACGATA AGGAGACCTGCTTTGCCGAGGAGGGTAAAAAACTTGTTGCTGCAAGTCAAGCTGCCTTA GGCTTAGGCGGAGGCGGTAGCGGAGGCGGTGGCTCCGGTGGCGGAGGGTCTGCTTACCG CCCCAGTGAGACCCTGTGCGGCGGGGAGCTGGTGGACACCCTCCAGTTCGTCTGTGGGG ACCGCGGCTTCTACTTCAGCAGGCCCGCAAGCCGTGTGAGCGcTCGCAGCCGTGGCATCG TTGAGGAGTGCTGTTTCCGCAGCTGTGACCTGGCCCTCCTGGAGACGTACTGTGCTACCC CCGCCAAGTCCGAG 44 6xHis-His tagged HSA fusion CACCATCACCATCACCATAGCGGCGATGC HSA-IGF2 R61Q mutant with ACACAAGAGTGAGGTTGCTCATCGGTTTA linker3-a long linker  AAGATTTGGGAGAAGAAAATTTCAAAGCC IGF2R61Qnucleotide sequence TTGGTGTTGATTGCCTTTGCTCAGTATCTTCAGCAGTGTCCATTTGAAGATCATGTAAAAT TAGTGAATGAAGTAACTGAATTTGCAAAAACATGTGTTGCTGATGAGTCAGCTGAAAAT TGTGACAAATCACTTCATACCCTTTTTGGAGACAAATTATGCACAGTTGCAACTCTTCGT GAAACCTATGGTGAAATGGCTGACTGCTGTGCAAAACAAGAACCTGAGAGAAATGAAT GCTTCTTGCAACACAAAGATGACAACCCAAACCTCCCCCGATTGGTGAGACCAGAGGT TGATGTGATGTGCACTGCTTTTCATGACAATGAAGAGACATTTTTGAAAAAATACTTATA TGAAATTGCCAGAAGACATCCTTACTTTTATGCCCCGGAACTCCTTTTCTTTGCTAAAAG GTATAAAGCTGCTTTTACAGAATGTTGCCAAGCTGCTGATAAAGCTGCCTGCCTGTTGCC AAAGCTCGATGAACTTCGGGATGAAGGGAAGGCTTCGTCTGCCAAACAGAGACTCAAG TGTGCCAGTCTCCAAAAATTTGGAGAAAGAGCTTTCAAAGCATGGGCAGTAGCTCGCCT GAGCCAGAGATTTCCCAAAGCTGAGTTTGCAGAAGTTTCCAAGTTAGTGACAGATCTTA CCAAAGTCCACACGGAATGCTGCCATGGAGATCTGCTTGAATGTGCTGATGACAGGGC GGACCTTGCCAAGTATATCTGTGAAAATCAAGATTCGATCTCCAGTAAACTGAAGGAAT GCTGTGAAAAACCTCTGTTGGAAAAATCCCACTGCATTGCCGAAGTGGAAAATGATGA GATGCCTGCTGACTTGCCTTCATTAGCTGCTGATTTTGTTGAAAGTAAGGATGTTTGCAA AAACTATGCTGAGGCAAAGGATGTCTTCCTGGGCATGTTTTTGTATGAATATGCAAGAAG GCATCCTGATTACTCTGTCGTGCTGCTGCTGAGACTTGCCAAGACATATGAAACCACTC TAGAGAAGTGCTGTGCCGCTGCAGATCCTCATGAATGCTATGCCAAAGTGTTCGATGAAT TTAAACCTCTTGTGGAAGAGCCTCAGAATTTAATCAAACAAAATTGTGAGCTTTTTGAGC AGCTTGGAGAGTACAAATTCCAGAATGCGCTATTAGTTCGTTACACCAAGAAAGTACCC CAAGTGTCAACTCCAACTCTTGTAGAGGTCTCAAGAAACCTAGGAAAAGTGGGCAGCAA ATGTTGTAAACATCCTGAAGCAAAAAGAATGCCCTGTGCAGAAGACTATCTATCCGTGG TCCTGAACCAGTTATGTGTGTTGCATGAGAAAACGCCAGTAAGTGACAGAGTCACCAAA TGCTGCACAGAATCCTTGGTGAACAGGCGACCATGCTTTTCAGCTCTGGAAGTCGATGA AACATACGTTCCCAAAGAGTTTAATGCTGAAACATTCACCTTCCATGCAGATATATGCAC ACTTTCTGAGAAGGAGAGACAAATCAAGAAACAAACTGCACTTGTTGAGCTCGTGAAA CACAAGCCCAAGGCAACAAAAGAGCAACTGAAAGCTGTTATGGATGATTTCGCAGCTTT TGTAGAGAAGTGCTGCAAGGCTGACGATAAGGAGACCTGCTTTGCCGAGGAGGGTAAA AAACTTGTTGCTGCAAGTCAAGCTGCCTTAGGCTTAGGCGGAGGCGGTAGCGGAGGCGG TGGCTCCGGTGGCGGAGGGTCTGCTTACCGCCCCAGTGAGACCCTGTGCGGCGGGGAGC TGGTGGACACCCTCCAGTTCGTCTGTGGGGACCGCGGCTTCTACTTCAGCAGGCCCGCAA GCCGTGTGAGCCaGCGCAGCCGTGGCATCGTTGAGGAGTGCTGTTTCCGCAGCTGTGACC TGGCCCTCCTGGAGACGTACTGTGCTACCCCCGCCAAGTCCGAG 45 6xHis- His tagged HAS fusionCACCATCACCATCACCATAGCGGCGATGC HSA- IGF2R64 A mutant withACACAAGAGTGAGGTTGCTCATCGGTTTA linker3- a long linker AAGATTTGGGAGAAGAAAATTTCAAAGCC IGF2R64A nucleotide sequenceTTGGTGTTGATTGCCTTTGCTCAGTATCTTC AGCAGTGTCCATTTGAAGATCATGTAAAATTAGTGAATGAAGTAACTGAATTTGCAAAA ACATGTGTTGCTGATGAGTCAGCTGAAAATTGTGACAAATCACTTCATACCCTTTTTGGA GACAAATTATGCACAGTTGCAACTCTTCGTGAAACCTATGGTGAAATGGCTGACTGCTG TGCAAAACAAGAACCTGAGAGAAATGAATGCTTCTTGCAACACAAAGATGACAACCCA AACCTCCCCCGATTGGTGAGACCAGAGGTTGATGTGATGTGCACTGCTTTTCATGACAA TGAAGAGACATTTTTGAAAAAATACTTATATGAAATTGCCAGAAGACATCCTTACTTTTA TGCCCCGGAACTCCTTTTCTTTGCTAAAAGGTATAAAGCTGCTTTTACAGAATGTTGCCA AGCTGCTGATAAAGCTGCCTGCCTGTTGCCAAAGCTCGATGAACTTCGGGATGAAGGGA AGGCTTCGTCTGCCAAACAGAGACTCAAGTGTGCCAGTCTCCAAAAATTTGGAGAAAG AGCTTTCAAAGCATGGGCAGTAGCTCGCCTGAGCCAGAGATTTCCCAAAGCTGAGTTTG CAGAAGTTTCCAAGTTAGTGACAGATCTTACCAAAGTCCACACGGAATGCTGCCATGGA GATCTGCTTGAATGTGCTGATGACAGGGCGGACCTTGCCAAGTATATCTGTGAAAATCA AGATTCGATCTCCAGTAAACTGAAGGAATGCTGTGAAAAACCTCTGTTGGAAAAATCC CACTGCATTGCCGAAGTGGAAAATGATGAGATGCCTGCTGACTTGCCTTCATTAGCTGC TGATTTTGTTGAAAGTAAGGATGTTTGCAAAAACTATGCTGAGGCAAAGGATGTCTTCCT GGGCATGTTTTTGTATGAATATGCAAGAAGGCATCCTGATTACTCTGTCGTGCTGCTGCT GAGACTTGCCAAGACATATGAAACCACTCTAGAGAAGTGCTGTGCCGCTGCAGATCCTC ATGAATGCTATGCCAAAGTGTTCGATGAATTTAAACCTCTTGTGGAAGAGCCTCAGAATT TAATCAAACAAAATTGTGAGCTTTTTGAGCAGCTTGGAGAGTACAAATTCCAGAATGCG CTATTAGTTCGTTACACCAAGAAAGTACCCCAAGTGTCAACTCCAACTCTTGTAGAGGTC TCAAGAAACCTAGGAAAAGTGGGCAGCAAATGTTGTAAACATCCTGAAGCAAAAAGAA TGCCCTGTGCAGAAGACTATCTATCCGTGGTCCTGAACCAGTTATGTGTGTTGCATGAGA AAACGCCAGTAAGTGACAGAGTCACCAAATGCTGCACAGAATCCTTGGTGAACAGGCG ACCATGCTTTTCAGCTCTGGAAGTCGATGAAACATACGTTCCCAAAGAGTTTAATGCTGA AACATTCACCTTCCATGCAGATATATGCACACTTTCTGAGAAGGAGAGACAAATCAAGA AACAAACTGCACTTGTTGAGCTCGTGAAACACAAGCCCAAGGCAACAAAAGAGCAACT GAAAGCTGTTATGGATGATTTCGCAGCTTTTGTAGAGAAGTGCTGCAAGGCTGACGATA AGGAGACCTGCTTTGCCGAGGAGGGTAAAAAACTTGTTGCTGCAAGTCAAGCTGCCTTA GGCTTAGGCGGAGGCGGTAGCGGAGGCGGTGGCTCCGGTGGCGGAGGGTCTGCTTACCG CCCCAGTGAGACCCTGTGCGGCGGGGAGCTGGTGGACACCCTCCAGTTCGTCTGTGGGG ACCGCGGCTTCTACTTCAGCAGGCCCGCAAGCCGTGTGAGCCGTCGCAGCGCTGGCATC GTTGAGGAGTGCTGTTTCCGCAGCTGTGACCTGGCCCTCCTGGAGACGTACTGTGCTACC CCCGCCAAGTCCGAG 46 6xHis-His tagged HAS fusion CACCATCACCATCACCATAGCGGCGATGC HSA-IGF2R64Q Mutant with ACACAAGAGTGAGGTTGCTCATCGGTTTA linker3-along linker  AAGATTTGGGAGAAGAAAATTTCAAAGCC IGF2R64Q nucleotide sequenceTTGGTGTTGATTGCCTTTGCTCAGTATCTTC AGCAGTGTCCATTTGAAGATCATGTAAAATTAGTGAATGAAGTAACTGAATTTGCAAAA ACATGTGTTGCTGATGAGTCAGCTGAAAATTGTGACAAATCACTTCATACCCTTTTTGGA GACAAATTATGCACAGTTGCAACTCTTCGTGAAACCTATGGTGAAATGGCTGACTGCTG TGCAAAACAAGAACCTGAGAGAAATGAATGCTTCTTGCAACACAAAGATGACAACCCA AACCTCCCCCGATTGGTGAGACCAGAGGTTGATGTGATGTGCACTGCTTTTCATGACAA TGAAGAGACATTTTTGAAAAAATACTTATATGAAATTGCCAGAAGACATCCTTACTTTTA TGCCCCGGAACTCCTTTTCTTTGCTAAAAGGTATAAAGCTGCTTTTACAGAATGTTGCCA AGCTGCTGATAAAGCTGCCTGCCTGTTGCCAAAGCTCGATGAACTTCGGGATGAAGGGA AGGCTTCGTCTGCCAAACAGAGACTCAAGTGTGCCAGTCTCCAAAAATTTGGAGAAAG AGCTTTCAAAGCATGGGCAGTAGCTCGCCTGAGCCAGAGATTTCCCAAAGCTGAGTTTG CAGAAGTTTCCAAGTTAGTGACAGATCTTACCAAAGTCCACACGGAATGCTGCCATGGA GATCTGCTTGAATGTGCTGATGACAGGGCGGACCTTGCCAAGTATATCTGTGAAAATCA AGATTCGATCTCCAGTAAACTGAAGGAATGCTGTGAAAAACCTCTGTTGGAAAAATCC CACTGCATTGCCGAAGTGGAAAATGATGAGATGCCTGCTGACTTGCCTTCATTAGCTGC TGATTTTGTTGAAAGTAAGGATGTTTGCAAAAACTATGCTGAGGCAAAGGATGTCTTCCT GGGCATGTTTTTGTATGAATATGCAAGAAGGCATCCTGATTACTCTGTCGTGCTGCTGCT GAGACTTGCCAAGACATATGAAACCACTCTAGAGAAGTGCTGTGCCGCTGCAGATCCTC ATGAATGCTATGCCAAAGTGTTCGATGAATTTAAACCTCTTGTGGAAGAGCCTCAGAATT TAATCAAACAAAATTGTGAGCTTTTTGAGCAGCTTGGAGAGTACAAATTCCAGAATGCG CTATTAGTTCGTTACACCAAGAAAGTACCCCAAGTGTCAACTCCAACTCTTGTAGAGGTC TCAAGAAACCTAGGAAAAGTGGGCAGCAAATGTTGTAAACATCCTGAAGCAAAAAGAA TGCCCTGTGCAGAAGACTATCTATCCGTGGTCCTGAACCAGTTATGTGTGTTGCATGAGA AAACGCCAGTAAGTGACAGAGTCACCAAATGCTGCACAGAATCCTTGGTGAACAGGCG ACCATGCTTTTCAGCTCTGGAAGTCGATGAAACATACGTTCCCAAAGAGTTTAATGCTGA AACATTCACCTTCCATGCAGATATATGCACACTTTCTGAGAAGGAGAGACAAATCAAGA AACAAACTGCACTTGTTGAGCTCGTGAAACACAAGCCCAAGGCAACAAAAGAGCAACT GAAAGCTGTTATGGATGATTTCGCAGCTTTTGTAGAGAAGTGCTGCAAGGCTGACGATA AGGAGACCTGCTTTGCCGAGGAGGGTAAAAAACTTGTTGCTGCAAGTCAAGCTGCCTTA GGCTTAGGCGGAGGCGGTAGCGGAGGCGGTGGCTCCGGTGGCGGAGGGTCTGCTTACCG CCCCAGTGAGACCCTGTGCGGCGGGGAGCTGGTGGACACCCTCCAGTTCGTCTGTGGGG ACCGCGGCTTCTACTTCAGCAGGCCCGCAAGCCGTGTGAGCCGTCGCAGCCaGGGCATCG TTGAGGAGTGCTGTTTCCGCAGCTGTGACCTGGCCCTCCTGGAGACGTACTGTGCTACCC CCGCCAAGTCCGAG 47 IGF2-Human IGF2- linker3- GCTTACCGCCCCAGTGAGACCCTGTGCGGC linker3-hFc4 nucleotide GGGGAGCTGGTGGACACCCTCCAGTTCGT hFc4 sequenceCTGTGGGGACCGCGGCTTCTACTTCAGCAG GCCCGCAAGCCGTGTGAGCCGTCGCAGCCGTGGCATCGTTGAGGAGTGCTGTTTCCGCA GCTGTGACCTGGCCCTCCTGGAGACGTACTGTGCTACCCCCGCCAAGTCCGAGGGCGGA GGCGGTAGCGGAGGCGGTGGCTCCGGTGGCGGAGGGTCTGAGTCCAAATATGGTCCCC CATGCCCACCCTGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCC CAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTG GACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGG TGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGT CAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGG TCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAG CCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACC AGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGG GAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCG ACGGCTCCTTCTTCCTCTACAGCAGGCTCACCGTGGACAAGAGCAGGTGGCAGGAGGGG AATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAG CCTCTCCCTGTCTCTGGGTAAA 48 IGF2-hFc4Human IGF2-hFc4 GCTTACCGCCCCAGTGAGACCCTGTGCGGC nucleotide sequenceGGGGAGCTGGTGGACACCCTCCAGTTCGT CTGTGGGGACCGCGGCTTCTACTTCAGCAGGCCCGCAAGCCGTGTGAGCCGTCGCAGCC GTGGCATCGTTGAGGAGTGCTGTTTCCGCAGCTGTGACCTGGCCCTCCTGGAGACGTACT GTGCTACCCCCGCCAAGTCCGAGGAGTCCAAATATGGTCCCCCATGCCCACCCTGCCCA GCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACAC TCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAG ACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGAC AAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCC TGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCT CCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGG TGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTG CCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGC CGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTC TACAGCAGGCTCACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTC CGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGG GTAAA 49 hFc4- hFc4- linker3- humanGAGTCCAAATATGGTCCCCCATGCCCACCC linker3- IGF2 nucleotideTGCCCAGCACCTGAGTTCCTGGGGGGACC IGF2 sequenceATCAGTCTTCCTGTTCCCCCCAAAACCCAA GGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCC AGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGC CAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCA CCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAA AGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGC CACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTG ACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGG GCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCT TCCTCTACAGCAGGCTCACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTC ATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTC TCTGGGTAAAGGCGGAGGCGGTAGCGGAGGCGGTGGCTCCGGTGGCGGAGGGTCTGCT TACCGCCCCAGTGAGACCCTGTGCGGCGGGGAGCTGGTGGACACCCTCCAGTTCGTCTG TGGGGACCGCGGCTTCTACTTCAGCAGGCCCGCAAGCCGTGTGAGCCGTCGCAGCCGTG GCATCGTTGAGGAGTGCTGTTTCCGCAGCTGTGACCTGGCCCTCCTGGAGACGTACTGTG CTACCCCCGCCAAGTCCGAG 50 IGF2R61A-Human IGF2 R61A GCTTACCGCCCCAGTGAGACCCTGTGCGGC linker3-point mutant- linker3- GGGGAGCTGGTGGACACCCTCCAGTTCGT hFc4hFc4 nucleotide CTGTGGGGACCGCGGCTTCTACTTCAGCAG sequenceGCCCGCAAGCCGTGTGAGCGcTCGCAGCCG TGGCATCGTTGAGGAGTGCTGTTTCCGCAGCTGTGACCTGGCCCTCCTGGAGACGTACTG TGCTACCCCCGCCAAGTCCGAGGGCGGAGGCGGTAGCGGAGGCGGTGGCTCCGGTGGC GGAGGGTCTGAGTCCAAATATGGTCCCCCATGCCCACCCTGCCCAGCACCTGAGTTCCT GGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCC GGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCA GTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAG GAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTG GCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCG AGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCC CCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGC TTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTA CAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTCA CCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAG GCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAA 51 BMP-7 Mature form of humanTCCACGGGGAGCAAACAGCGCAGCCAGAA BMP-7 (AA293-431)CCGCTCCAAGACGCCCAAGAACCAGGAAG nucleotide sequenceCCCTGCGGATGGCCAACGTGGCAGAGAAC AGCAGCAGCGACCAGAGGCAGGCCTGTAAGAAGCACGAGCTGTATGTCAGCTTCCGAG ACCTGGGCTGGCAGGACTGGATCATCGCGCCTGAAGGCTACGCCGCCTACTACTGTGAG GGGGAGTGTGCCTTCCCTCTGAACTCCTACATGAACGCCACCAACCACGCCATCGTGCA GACGCTGGTCCACTTCATCAACCCGGAAACGGTGCCCAAGCCCTGCTGTGCGCCCACGC AGCTCAATGCCATCTCCGTCCTCTACTTCGATGACAGCTCCAACGTCATCCTGAAGAAA TACAGAAACATGGTGGTCCGGGCCTGTGG CTGCCAC 52BMP-7- BMP7 fusion protein TCCACGGGGAGCAAACAGCGCAGCCAGAA linker1-nucleotide sequence CCGCTCCAAGACGCCCAAGAACCAGGAAG hFcmCCCTGCGGATGGCCAACGTGGCAGAGAAC AGCAGCAGCGACCAGAGGCAGGCCTGTAAGAAGCACGAGCTGTATGTCAGCTTCCGAG ACCTGGGCTGGCAGGACTGGATCATCGCGCCTGAAGGCTACGCCGCCTACTACTGTGAG GGGGAGTGTGCCTTCCCTCTGAACTCCTACATGAACGCCACCAACCACGCCATCGTGCA GACGCTGGTCCACTTCATCAACCCGGAAACGGTGCCCAAGCCCTGCTGTGCGCCCACGC AGCTCAATGCCATCTCCGTCCTCTACTTCGATGACAGCTCCAACGTCATCCTGAAGAAA TACAGAAACATGGTGGTCCGGGCCTGTGGCTGCCACGGATCGGGATCGGACAAAACTC ACACATGCCCACCGTGCCCAGCACCTGAAGCTGCCGGGGGACCGTCAGTCTTCCTCTTC CCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGT GGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTG GAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTG TGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGC AAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAG GGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAA GAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGG AGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGA CTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGC AGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAG AAaAGCCTCTCCCTGTCTCCGGGTAAA 53 BMP-7-BMP7 fusion protein TCCACGGGGAGCAAACAGCGCAGCCAGAA linker2-nucleotide sequence CCGCTCCAAGACGCCCAAGAACCAGGAAG hFcmCCCTGCGGATGGCCAACGTGGCAGAGAAC AGCAGCAGCGACCAGAGGCAGGCCTGTAAGAAGCACGAGCTGTATGTCAGCTTCCGAG ACCTGGGCTGGCAGGACTGGATCATCGCGCCTGAAGGCTACGCCGCCTACTACTGTGAG GGGGAGTGTGCCTTCCCTCTGAACTCCTACATGAACGCCACCAACCACGCCATCGTGCA GACGCTGGTCCACTTCATCAACCCGGAAACGGTGCCCAAGCCCTGCTGTGCGCCCACGC AGCTCAATGCCATCTCCGTCCTCTACTTCGATGACAGCTCCAACGTCATCCTGAAGAAA TACAGAAACATGGTGGTCCGGGCCTGTGGCTGCCACGGATCTGGGAGCGCTGACAAAA CTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCCGGGGGACCGTCAGTCTTCCTCT TCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTG GTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCG TGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCG TGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGT GCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAA AGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCA AGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTG GAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTG GACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCA GCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGC AGAAaAGCCTCTCCCTGTCTCCGGGTAAA 54 FGF-17Full length of human TQGENHPSPNFNQYVRDQGAMTDQLSRRQI FGF-17 amino acidREYQLYSRTSGKHVQVTGRRISATAEDGNKF sequence AKLIVETDTFGSRVRIKGAESEKYICMNKRGKLIGKPSGKSKDCVFTEIVLENNYTAFQNAR HEGWFMAFTRQGRPRQASRSRQNQREAHFIKRLYQGQLPFPNHAEKQKQFEFVGSAPTRRT KRTRRPQPLT 55 FGF- Human FGF-17 AA204-TQGENHPSPNFNQYVRDQGAMTDQLSRRQI 17d204- 216 deletion mutantREYQLYSRTSGKHVQVTGRRISATAEDGNKF 216 amino acid sequenceAKLIVETDTFGSRVRIKGAESEKYICMNKRG KLIGKPSGKSKDCVFTEIVLENNYTAFQNARHEGWFMAFTRQGRPRQASRSRQNQREAHFI KRLYQGQLPFPNHAEKQKQFEFVGSAPT 56 FGF-Human FGF-17 AA181- TQGENHPSPNFNQYVRDQGAMTDQLSRRQI 17d181-216 deletion mutant REYQLYSRTSGKHVQVTGRRISATAEDGNKF  216amino acid sequence AKLIVETDTFGSRVRIKGAESEKYICMNKRGKLIGKPSGKSKDCVFTEIVLENNYTAFQNAR HEGWFMAFTRQGRPRQASRSRQNQREAHFI KRLYQ 57FGF- Human FGF-17 R204Q TQGENHPSPNFNQYVRDQGAMTDQLSRRQI 17R204QK207Q mutant amino REYQLYSRTSGKHVQVTGRRISATAEDGNKF K207Q acid sequenceAKLIVETDTFGSRVRIKGAESEKYICMNKRG KLIGKPSGKSKDCVFTEIVLENNYTAFQNARHEGWFMAFTRQGRPRQASRSRQNQREAHFI KRLYQGQLPFPNHAEKQKQFEFVGSAPTQRTQRTRRPQPLT 58 FGF- Human FGF-17 AA197- TQGENHPSPNFNQYVRDQGAMTDQLSRRQI17d197- 216 deletion mutant REYQLYSRTSGKHVQVTGRRISATAEDGNKF 216amino acid sequence AKLIVETDTFGSRVRIKGAESEKYICMNKRGKLIGKPSGKSKDCVFTEIVLENNYTAFQNAR HEGWFMAFTRQGRPRQASRSRQNQREAHFIKRLYQGQLPFPNHAEKQKQFE 59 FGF- Human FGF-17 K191ATQGENHPSPNFNQYVRDQGAMTDQLSRRQI 17K191A K193A S200A mutantREYQLYSRTSGKHVQVTGRRISATAEDGNKF K193A amino acid sequenceAKLIVETDTFGSRVRIKGAESEKYICMNKRG S200A KLIGKPSGKSKDCVFTEIVLENNYTAFQNARHEGWFMAFTRQGRPRQASRSRQNQREAHFI KRLYQGQLPFPNHAEAQAQFEFVGAAPTRRTKRTRRPQPLT 60 FGF-17- Full length of humanTQGENHPSPNFNQYVRDQGAMTDQLSRRQI linker1- FGF-17- linker1- hFcmREYQLYSRTSGKHVQVTGRRISATAEDGNKF hFcm amino acid sequenceAKLIVETDTFGSRVRIKGAESEKYICMNKRG KLIGKPSGKSKDCVFTEIVLENNYTAFQNARHEGWFMAFTRQGRPRQASRSRQNQREAHFI KRLYQGQLPFPNHAEKQKQFEFVGSAPTRRTKRTRRPQPLTGSGSDKTHTCPPCPAPEAAGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK 61 FGF-Human FGF-17 AA204- TQGENHPSPNFNQYVRDQGAMTDQLSRRQI 17d204-216 deletion mutant- REYQLYSRTSGKHVQVTGRRISATAEDGNKF 216-linker1- hFcm amino AKLIVETDTFGSRVRIKGAESEKYICMNKRG linker1-acid sequence KLIGKPSGKSKDCVFTEIVLENNYTAFQNAR hFcmHEGWFMAFTRQGRPRQASRSRQNQREAHFI KRLYQGQLPFPNHAEKQKQFEFVGSAPTGSGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK 62 FGF-Human FGF-17 AA181- TQGENHPSPNFNQYVRDQGAMTDQLSRRQI 17d181-216 deletion mutant- REYQLYSRTSGKHVQVTGRRISATAEDGNKF 216-linker1- hFcm amino AKLIVETDTFGSRVRIKGAESEKYICMNKRG linker1-acid sequence KLIGKPSGKSKDCVFTEIVLENNYTAFQNAR hFcmHEGWFMAFTRQGRPRQASRSRQNQREAHFI KRLYQGSGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPI EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 63 FGF- Human FGF-17 R204QTQGENHPSPNFNQYVRDQGAMTDQLSRRQI 17R204Q K207Q mutant- linker1-REYQLYSRTSGKHVQVTGRRISATAEDGNKF K207Q- hFcm amino acidAKLIVETDTFGSRVRIKGAESEKYICMNKRG linker1- sequenceKLIGKPSGKSKDCVFTEIVLENNYTAFQNAR hFcm HEGWFMAFTRQGRPRQASRSRQNQREAHFIKRLYQGQLPFPNHAEKQKQFEFVGSAPTQRT QRTRRPQPLTGSGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 64 FGF- Human FGF17 AA197-TQGENHPSPNFNQYVRDQGAMTDQLSRRQI 17d197- 216 deletion mutant-REYQLYSRTSGKHVQVTGRRISATAEDGNKF 216- linker1- hFcmAKLIVETDTFGSRVRIKGAESEKYICMNKRG linker1- KLIGKPSGKSKDCVFTEIVLENNYTAFQNARhFcm HEGWFMAFTRQGRPRQASRSRQNQREAHFI KRLYQGQLPFPNHAEKQKQFEGSGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK 65 FGF- Human FGF-17TQGENHPSPNFNQYVRDQGAMTDQLSRRQI 17K191A K191A K193AREYQLYSRTSGKHVQVTGRRISATAEDGNKF K193A S200A mutant-AKLIVETDTFGSRVRIKGAESEKYICMNKRG S200A- linker1-hFcmKLIGKPSGKSKDCVFTEIVLENNYTAFQNAR linker1- HEGWFMAFTRQGRPRQASRSRQNQREAHFIhFcm KRLYQGQLPFPNHAEAQAQFEFVGAAPTRR TKRTRRPQPLTGSGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 66 FGF-17- Full length ofTQGENHPSPNFNQYVRDQGAMTDQLSRRQI linker2- human FGF1REYQLYSRTSGKHVQVTGRRISATAEDGNKF  hFcm 7-linker2-hFcmAKLIVETDTFGSRVRIKGAESEKYICMNKRG KLIGKPSGKSKDCVFTEIVLENNYTAFQNARHEGWFMAFTRQGRPRQASRSRQNQREAHFI KRLYQGQLPFPNHAEKQKQFEFVGSAPTRRTKRTRRPQPLTGSGSADKTHTCPPCPAPEAAG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDEL TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK 67 FGF-Human FGF17 AA204- TQGENHPSPNFNQYVRDQGAMTDQLSRRQI 17d204-216 deletion mutant- REYQLYSRTSGKHVQVTGRRISATAEDGNKF 216- linker2- hFcmAKLIVETDTFGSRVRIKGAESEKYICMNKRG linker2- KLIGKPSGKSKDCVFTEIVLENNYTAFQNARhFcm HEGWFMAFTRQGRPRQASRSRQNQREAHFI KRLYQGQLPFPNHAEKQKQFEFVGSAPTGSGSADKTHTCPPCPAPEAAGGPSVFLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK 68 FGF-Human FGF17 AA181- TQGENHPSPNFNQYVRDQGAMTDQLSRRQI 17d181-216 deletion mutant- REYQLYSRTSGKHVQVTGRRISATAEDGNKF 216- linker2- hFcmAKLIVETDTFGSRVRIKGAESEKYICMNKRG linker2- KLIGKPSGKSKDCVFTEIVLENNYTAFQNARhFcm HEGWFMAFTRQGRPRQASRSRQNQREAHFI KRLYQGSGSADKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGA PIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 69 FGF- Human FGF-17 R204QTQGENHPSPNFNQYVRDQGAMTDQLSRRQI 17R204Q K207Q mutant- linker2-REYQLYSRTSGKHVQVTGRRISATAEDGNKF K207Q- hFcmAKLIVETDTFGSRVRIKGAESEKYICMNKRG linker2- KLIGKPSGKSKDCVFTEIVLENNYTAFQNARhFcm HEGWFMAFTRQGRPRQASRSRQNQREAHFI KRLYQGQLPFPNHAEKQKQFEFVGSAPTQRTQRTRRPQPLTGSGSADKTHTCPPCPAPEAAG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDEL TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK 70 FGF-Human FGF17 AA197- TQGENHPSPNFNQYVRDQGAMTDQLSRRQI 17d197-216 deletion mutant- REYQLYSRTSGKHVQVTGRRISATAEDGNKF 216- linker2- hFcmAKLIVETDTFGSRVRIKGAESEKYICMNKRG linker2- KLIGKPSGKSKDCVFTEIVLENNYTAFQNARhFcm HEGWFMAFTRQGRPRQASRSRQNQREAHFI KRLYQGQLPFPNHAEKQKQFEGSGSADKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRT PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK 71 FGF- Human FGF-17 K191ATQGENHPSPNFNQYVRDQGAMTDQLSRRQI 17K 191AK K193A S200A mutant-REYQLYSRTSGKHVQVTGRRISATAEDGNKF 193AS200 linker2- hFcmAKLIVETDTFGSRVRIKGAESEKYICMNKRG A-linker2-KLIGKPSGKSKDCVFTEIVLENNYTAFQNAR hFcm HEGWFMAFTRQGRPRQASRSRQNQREAHFIKRLYQGQLPFPNHAEAQAQFEFVGAAPTRR TKRTRRPQPLTGSGSADKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 72 6xHis- His tagged HSA fusionHHHHHHSGDAHKSEVAHRFKDLGEENFKAL HSA- FGF17 with a longVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTC linker3- linkerVADESAENCDKSLHTLFGDKLCTVATLRET FGF17 YGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIAR RHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQ KFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYI CENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVF LGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQN LIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMP CAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTF HADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFA EEGKKLVAASQAALGLGGGGSGGGGSGGGGSTQGENHPSPNFNQYVRDQGAMTDQLSRR QIREYQLYSRTSGKHVQVTGRRISATAEDGNKFAKLIVETDTFGSRVRIKGAESEKYICMNK RGKLIGKPSGKSKDCVFTEIVLENNYTAFQNARHEGWFMAFTRQGRPRQASRSRQNQREA HFIKRLYQGQLPFPNHAEKQKQFEFVGSAPTRRTKRTRRPQPLT 73 FGF17- Human FGF17-linker3-TQGENHPSPNFNQYVRDQGAMTDQLSRRQI linker3- hFc4REYQLYSRTSGKHVQVTGRRISATAEDGNKF hFc4 AKLIVETDTFGSRVRIKGAESEKYICMNKRGKLIGKPSGKSKDCVFTEIVLENNYTAFQNAR HEGWFMAFTRQGRPRQASRSRQNQREAHFIKRLYQGQLPFPNHAEKQKQFEFVGSAPTRRT KRTRRPQPLTGGGGSGGGGSGGGGSESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRT PEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYT QKSLSLSLGK 74 FGF17- Human FGF17-hFc4TQGENHPSPNFNQYVRDQGAMTDQLSRRQI hFc4 REYQLYSRTSGKHVQVTGRRISATAEDGNKFAKLIVETDTFGSRVRIKGAESEKYICMNKRG KLIGKPSGKSKDCVFTEIVLENNYTAFQNARHEGWFMAFTRQGRPRQASRSRQNQREAHFI KRLYQGQLPFPNHAEKQKQFEFVGSAPTRRTKRTRRPQPLTESKYGPPCPPCPAPEFLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFS CSVMHEALHNHYTQKSLSLSLGK 75 hFc4L-hFc4-linker3-human ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDT FGF17 FGF17LMISRTPEVTCVVVDVSQEDPEVQFNWYVD GVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKG QPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSGGGGST QGENHPSPNFNQYVRDQGAMTDQLSRRQIREYQLYSRTSGKHVQVTGRRISATAEDGNKF AKLIVETDTFGSRVRIKGAESEKYICMNKRGKLIGKPSGKSKDCVFTEIVLENNYTAFQNAR HEGWFMAFTRQGRPRQASRSRQNQREAHFIKRLYQGQLPFPNHAEKQKQFEFVGSAPTRRT KRTRRPQPLT 76 IGF2 human IGF2 amino acidAYRPSETLCGGELVDTLQFVCGDRGFYFSRP sequenceASRVSRRSRGIVEECCFRSCDLALLETYCATP AKSE 77 IGF2-AYRPSETLCGGELVDTLQFVCGDRGFYFSRP linker1-ASRVSRRSRGIVEECCFRSCDLALLETYCATP hFcm AKSEGSGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 78 IGF2- AYRPSETLCGGELVDTLQFVCGDRGFYFSRP linker2-ASRVSRRSRGIVEECCFRSCDLALLETYCATP hFcm AKSEGSGSADKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPI EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 79 IGF2Big Full-length human IGF2MGIPMGKSMLVLLTFLAFASCCIAAYRPSET LCGGELVDTLQFVCGDRGFYFSRPASRVSRRSRGIVEECCFRSCDLALLETYCATPAKSERD VSTPPTVLPDNFPRYPVGKFFQYDTWKQSTQRLRRGLPALLRARRGHVLAKELEAFREAKR HRPLIALPTQDPAHGGAPPEMASNRK 80 6xHis-His tagged HSA fusion HHHHHHSGDAHKSEVAHRFKDLGEENFKAL HSA-IGF2 with a long linker VLIAFAQYLQQCPFEDHVKLVNEVTEFAKTC linker3-VADESAENCDKSLHTLFGDKLCTVATLRET IGF2 YGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIAR RHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQ KFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYI CENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVF LGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQN LIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMP CAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTF HADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFA EEGKKLVAASQAALGLGGGGSGGGGSGGGGSAYRPSETLCGGELVDTLQFVCGDRGFYFS RPASRVSRRSRGIVEECCFRSCDLALLETYCA TPAKSE81 6xHis- His tagged HSA fusion HHHHHHSGDAHKSEVAHRFKDLGEENFKAL HSA-IGF2 R61A mutant with VLIAFAQYLQQCPFEDHVKLVNEVTEFAKTC linker3-a long linker VADESAENCDKSLHTLFGDKLCTVATLRET IGF2R61AYGEMADCCAKQEPERNECFLQHKDDNPNLP RLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADK AACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKL VTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVEND EMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETT LEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQ VSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCC TESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKAT KEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGGGGSGGGGSGGG GSAYRPSETLCGGELVDTLQFVCGDRGFYFSRPASRVSARSRGIVEECCFRSCDLALLETYCA TPAKSE 82 6xHis- His tagged HSA fusionHHHHHHSGDAHKSEVAHRFKDLGEENFKAL HSA- IGF2 R61Q mutant withVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTC linker3- a long linkerVADESAENCDKSLHTLFGDKLCTVATLRET IGF2R61Q YGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIAR RHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQ KFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYI CENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVF LGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQN LIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMP CAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTF HADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFA EEGKKLVAASQAALGLGGGGSGGGGSGGGGSAYRPSETLCGGELVDTLQFVCGDRGFYFS RPASRVSQRSRGIVEECCFRSCDLALLETYCA TPAKSE83 6xHis- His tagged HSA fusion HHHHHHSGDAHKSEVAHRFKDLGEENFKAL HSA-IGF2 R64A mutant with VLIAFAQYLQQCPFEDHVKLVNEVTEFAKTC linker3-a long linker VADESAENCDKSLHTLFGDKLCTVATLRET IGF2R64AYGEMADCCAKQEPERNECFLQHKDDNPNLP RLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADK AACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKL VTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVEND EMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETT LEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQ VSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCC TESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKAT KEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGGGGSGGGGSGGG GSAYRPSETLCGGELVDTLQFVCGDRGFYFSRPASRVSRRSAGIVEECCFRSCDLALLETYCA TPAKSE 84 6xHis- His tagged HSA fusionHHHHHHSGDAHKSEVAHRFKDLGEENFKAL HSA- IGF2 R64Q mutant withVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTC linker3- a long linkerVADESAENCDKSLHTLFGDKLCTVATLRET IGF2R64Q YGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIAR RHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQ KFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYI CENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVF LGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQN LIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMP CAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTF HADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFA EEGKKLVAASQAALGLGGGGSGGGGSGGGGSAYRPSETLCGGELVDTLQFVCGDRGFYFS RPASRVSRRSQGIVEECCFRSCDLALLETYCA TPAKSE85 IGF2- Human IGF2-linker3- AYRPSETLCGGELVDTLQFVCGDRGFYFSRP linker3-hFc4 ASRVSRRSRGIVEECCFRSCDLALLETYCATP hFc4AKSEGGGGSGGGGSGGGGSESKYGPPCPPCP APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKP REEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPP SQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKS RWQEGNVFSCSVMHEALHNHYTQKSLSLSL GK 86IGF2-hFc4 HumanIGF2-hFc4 AYRPSETLCGGELVDTLQFVCGDRGFYFSRPASRVSRRSRGIVEECCFRSCDLALLETYCATP AKSEESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFN WYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS KAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 87 hFc4- hFc4-linker3-humanESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDT linker3- IGF2LMISRTPEVTCVVVDVSQEDPEVQFNWYVD IGF2 GVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKG QPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSGGGGSA YRPSETLCGGELVDTLQFVCGDRGFYFSRPASRVSRRSRGIVEECCFRSCDLALLETYCATPA KSE 88 IGF2R61A- Human IGF2 R61AAYRPSETLCGGELVDTLQFVCGDRGFYFSRP linker3- point mutant- linker3-ASRVSARSRGIVEECCFRSCDLALLETYCATP hFc4 hFc4AKSEGGGGSGGGGSGGGGSESKYGPPCPPCP APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKP REEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPP SQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKS RWQEGNVFSCSVMHEALHNHYTQKSLSLSL GK 89BMP-7 Mature form of human STGSKQRSQNRSKTPKNQEALRMANVAENSBMP-7 (AA293-431) SSDQRQACKKHELYVSFRDLGWQDWIIAPEGYAAYYCEGECAFPLNSYMNATNHAIVQTL VHFINPETVPKPCCAPTQLNAISVLYFDDSSNVILKKYRNMVVRACGCH 90 BMP-7- BMP7 fusion proteinSTGSKQRSQNRSKTPKNQEALRMANVAENS linker1- SSDQRQACKKHELYVSFRDLGWQDWIIAPEhFcm GYAAYYCEGECAFPLNSYMNATNHAIVQTL VHFINPETVPKPCCAPTQLNAISVLYFDDSSNVILKKYRNMVVRACGCHGSGSDKTHTCPPC PAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK 91BMP-7- BMP7 fusion protein STGSKQRSQNRSKTPKNQEALRMANVAENS linker2-SSDQRQACKKHELYVSFRDLGWQDWIIAPE hFcm GYAAYYCEGECAFPLNSYMNATNHAIVQTLVHFINPETVPKPCCAPTQLNAISVLYFDDSSN VILKKYRNMVVRACGCHGSGSADKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK 92 BMP7 BMP7knuckleACGGTGCCCAAGCCCTGCTGTGCGCCCAC GCAGCTCAATGCCATCTCCGTCCTCTACTTCGATGACAGCTCCAACGTCATCCTGAAGA AATACAGA 93 BMP7 BMP7 KnuckleTVPKPCCAPTQLNAISVLYFDDSSNVILKKYR [A1][A2]

TABLE 3 Linker and Heterologous Sequences Seq ID Sequence No NameDescription Sequence 94 Linker1 A short flexible linker GGATCGGGATCGnucleotide sequence 95 Linker2 A short flexible linker GGATCTGGGAGCGCTnucleotide sequence 96 Linker3 A long flexible linkerGGCGGAGGCGGTAGCGGAGGCGGTGGCTC nucleotide sequence CGGTGGCGGAGGGTCT 97hFcmIgG1 HumanIgG1Fcmutant GACAAAACTCACACATGCCCACCGTGCCC(L234AL235AP329G) AGCACCTGAAGCTGCCGGGGGACCGTCAG nucleotide sequenceTCTTCCTCTTCCCCCCAAAACCCAAGGACA CCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAA GACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGA CAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTC CTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCC TCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACA GGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCT GCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAG CCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCT CTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGC TCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAaAGCCTCTCCCTGTCTCCG GGTAAA 98 hFc4 Human IgG4 Fc withGAGTCCAAATATGGTCCCCCATGCCCACCC S228P point mutationTGCCCAGCACCTGAGTTCCTGGGGGGACC nucleotide sequenceATCAGTCTTCCTGTTCCCCCCAAAACCCAA GGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCC AGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGC CAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCA CCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAA AGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGC CACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTG ACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGG GCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCT TCCTCTACAGCAGGCTCACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTC ATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAA GAGCCTCTCCCTGTCTCTGGGTAAA 99 6xHis Six Histidine shortCACCATCACCATCACCAT peptide nucleotide sequence 100 Strep IIStrep-Tactin binding TGGAGCCACCCGCAGTTCGAAAAA peptide nucleotidesequence 101 HSA Full length of Human GATGCACACAAGAGTGAGGTTGCTCATCGSerum Albumin GTTTAAAGATTTGGGAGAAGAAAATTTCA nucleotide sequenceAAGCCTTGGTGTTGATTGCCTTTGCTCAGT ATCTTCAGCAGTGTCCATTTGAAGATCATGTAAAATTAGTGAATGAAGTAACTGAATTT GCAAAAACATGTGTTGCTGATGAGTCAGCTGAAAATTGTGACAAATCACTTCATACCCT TTTTGGAGACAAATTATGCACAGTTGCAACTCTTCGTGAAACCTATGGTGAAATGGCTGA CTGCTGTGCAAAACAAGAACCTGAGAGAAATGAATGCTTCTTGCAACACAAAGATGAC AACCCAAACCTCCCCCGATTGGTGAGACCAGAGGTTGATGTGATGTGCACTGCTTTTCA TGACAATGAAGAGACATTTTTGAAAAAATACTTATATGAAATTGCCAGAAGACATCCTT ACTTTTATGCCCCGGAACTCCTTTTCTTTGCTAAAAGGTATAAAGCTGCTTTTACAGAAT GTTGCCAAGCTGCTGATAAAGCTGCCTGCCTGTTGCCAAAGCTCGATGAACTTCGGGATG AAGGGAAGGCTTCGTCTGCCAAACAGAGACTCAAGTGTGCCAGTCTCCAAAAATTTGGA GAAAGAGCTTTCAAAGCATGGGCAGTAGCTCGCCTGAGCCAGAGATTTCCCAAAGCTG AGTTTGCAGAAGTTTCCAAGTTAGTGACAGATCTTACCAAAGTCCACACGGAATGCTGCC ATGGAGATCTGCTTGAATGTGCTGATGACAGGGCGGACCTTGCCAAGTATATCTGTGAA AATCAAGATTCGATCTCCAGTAAACTGAAGGAATGCTGTGAAAAACCTCTGTTGGAAA AATCCCACTGCATTGCCGAAGTGGAAAATGATGAGATGCCTGCTGACTTGCCTTCATTA GCTGCTGATTTTGTTGAAAGTAAGGATGTTTGCAAAAACTATGCTGAGGCAAAGGATGT CTTCCTGGGCATGTTTTTGTATGAATATGCAAGAAGGCATCCTGATTACTCTGTCGTGCT GCTGCTGAGACTTGCCAAGACATATGAAACCACTCTAGAGAAGTGCTGTGCCGCTGCA GATCCTCATGAATGCTATGCCAAAGTGTTCGATGAATTTAAACCTCTTGTGGAAGAGCCT CAGAATTTAATCAAACAAAATTGTGAGCTTTTTGAGCAGCTTGGAGAGTACAAATTCCA GAATGCGCTATTAGTTCGTTACACCAAGAAAGTACCCCAAGTGTCAACTCCAACTCTTGT AGAGGTCTCAAGAAACCTAGGAAAAGTGGGCAGCAAATGTTGTAAACATCCTGAAGCA AAAAGAATGCCCTGTGCAGAAGACTATCTATCCGTGGTCCTGAACCAGTTATGTGTGTT GCATGAGAAAACGCCAGTAAGTGACAGAGTCACCAAATGCTGCACAGAATCCTTGGTGA ACAGGCGACCATGCTTTTCAGCTCTGGAAGTCGATGAAACATACGTTCCCAAAGAGTTTA ATGCTGAAACATTCACCTTCCATGCAGATATATGCACACTTTCTGAGAAGGAGAGACAA ATCAAGAAACAAACTGCACTTGTTGAGCTCGTGAAACACAAGCCCAAGGCAACAAAAG AGCAACTGAAAGCTGTTATGGATGATTTCGCAGCTTTTGTAGAGAAGTGCTGCAAGGCT GACGATAAGGAGACCTGCTTTGCCGAGGAGGGTAAAAAACTTGTTGCTGCAAGTCAAG CTGCCTTAGGCTTA 102 Linker1A short flexible linker GSGS amino acid sequence 103 Linker2A short flexible linker GSGSA amino acid sequence 104 Linker3A long flexible linker GGGGSGGGGSGGGGS amino acid sequence 105 hFcm IgG1HumanIgG1Fc mutant DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTL (L234A L235A P329G)MISRTPEVTCVVVDVSHEDPEVKFNWYVDG amino acid sequenceVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK 106 hFc4Human IgG4 Fc with ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDT S228P point mutationLMISRTPEVTCVVVDVSQEDPEVQFNWYVD amino acid sequenceGVEVHNAKTKPREEQFNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALH NHYTQKSLSLSLGK 107 6xHisSix Histidine short HHHHHH peptide amino acid sequence 108 Strep IIStrep-Tactin binding WSHPQFEK peptide amino acid sequence 109 HSAFull length of Human DAHKSEVAHRFKDLGEENFKALVLIAFAQY Serum Albumin aminoLQQCPFEDHVKLVNEVTEFAKTCVADESAE acid sequenceNCDKSLHTLFGDKLCTVATLRETYGEMADC CAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFY APELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERA FKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSI SSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLY EYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCE LFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLS VVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTL SEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKL VAASQAALGL

What is claimed is:
 1. A fusion protein, comprising: a human serumalbumin (HSA) polypeptide covalently coupled to an insulin-like growthfactor 2 (IGF2) polypeptide comprising an amino acid sequence at least90% identical to SEQ ID NO:
 76. 2. The fusion protein of claim 1,wherein the HSA polypeptide comprises an amino acid sequence at least90% identical to SEQ ID NO:
 109. 3. The fusion protein of claim 1,wherein the HSA polypeptide comprises the amino acid sequence of SEQ IDNO:
 109. 4. The fusion protein of claim 1, wherein the IGF2 polypeptideconsists of an amino acid sequence that is at least 90% identical to SEQID NO:
 76. 5. The fusion protein of claim 1, wherein the IGF2polypeptide amino acid sequence is at least 98% identical to SEQ ID NO:76.
 6. The fusion protein of claim 5, wherein the IGF2 polypeptideconsists of an amino acid sequence that is at least 98% identical to SEQID NO:
 76. 7. The fusion protein of claim 1, wherein the HSA polypeptideis fused to the IGF2 polypeptide at the N-terminus of the IGF2polypeptide sequence.
 8. The fusion protein of claim 1, wherein the HSApolypeptide is fused to the IGF2 polypeptide at the C-terminus of theIGF2 polypeptide sequence.
 9. The fusion protein of claim 1, wherein apeptide linker separates the HSA polypeptide and the IGF2 polypeptide inthe fusion protein.
 10. The fusion protein of claim 9, wherein thepeptide linker is covalently coupled to the C-terminus of the HSApolypeptide and to the N-terminus of the IGF2 polypeptide.
 11. Thefusion protein of claim 9, wherein the peptide linker is covalentlycoupled to the C-terminus of the IGF2 polypeptide and to the N-terminusof the HSA polypeptide.
 12. The fusion protein of claim 9, wherein thepeptide linker comprises a flexible peptide linker.
 13. The fusionprotein of claim 12, wherein the flexible peptide linker comprises aglycine-rich linker.
 14. The fusion protein of claim 13, wherein theglycine-rich linker comprises a glycine-serine linker.
 15. The fusionprotein of claim 14, wherein the glycine-serine linker comprises amultimer of the sequence GGGGS(n), wherein n is 1-5.
 16. The fusionprotein of claim 14, wherein the glycine-serine linker comprises theamino acid sequence of SEQ ID NO:
 104. 17. A fusion protein, comprising:a human serum albumin (HSA) polypeptide covalently coupled to aninsulin-like growth factor 2 (IGF2) polypeptide comprising an N-terminaldeletion or a C-terminal deletion.
 18. The fusion protein of claim 17,wherein the IGF2 polypeptide comprises the N-terminal deletion and theC-terminal deletion.
 19. A pharmaceutical composition comprising: afusion protein comprising: i. a human serum albumin (HSA) polypeptide,ii. an insulin-like growth factor 2 (IGF2) polypeptide comprising anamino acid sequence at least 90% identical to SEQ ID NO: 76, and iii. apeptide linker covalently coupled to the HSA polypeptide and to the IGF2polypeptide; and a pharmaceutically acceptable excipient, carrier, ordiluent.